Tuning cascade assay kinetics via molecular design

ABSTRACT

The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods allow one to control reaction kinetics of the cascade assay by two orders of magnitude via molecular design of one of the reaction components; further, varying molecular design also allows for quantification of target nucleic acids of interest over a large range of concentrations or discriminating between extremely low copy numbers of target nucleic acids of interest.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 18/076,262, filed 6Dec. 2022, which claims priority to U.S. Ser. No. 63/289,112, filed 13Dec. 2021; and U.S. Ser. No. 63/402,055, filed 29 Aug. 2022 all of whichare incorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

Submitted herewith is an electronically filed sequence listing viaEFS-Web a Sequence Listing XML, entitled “LS005US1_seqlist_20221206”,created 6 Dec. 2022, which is 4,769 bytes in size. The sequence listingis part of the specification of this specification and is incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to compositions of matter and assaymethods used to detect one or more target nucleic acids of interest in asample. The compositions and methods allow one to control reactionkinetics of a cascade assay by two orders of magnitude via the moleculardesign of one of the reaction components; further, varying moleculardesign also allows for quantification of target nucleic acids ofinterest over a large range of concentrations or discriminating betweencopy numbers of target nucleic acids of interest with exquisiteaccuracy.

BACKGROUND OF THE INVENTION

In the following discussion, certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Rapid and accurate identification of, e.g., infectious agents, microbecontamination, variant nucleic acid sequences that indicate the presenceof diseases such as cancer or contamination by heterologous sources isimportant in order to select correct treatment; identify tainted food,pharmaceuticals, cosmetics and other commercial goods; and to monitorthe environment including identification of biothreats. Classic PCR andnucleic acid-guided nuclease or CRISPR (clustered regularly interspacedshort palindromic repeats) detection methods rely on pre-amplificationof target nucleic acids of interest to enhance detection sensitivity.However, amplification increases time to detection and may cause changesto the relative proportion of nucleic acids in samples that, in turn,lead to artifacts or inaccurate results. Improved assays that allow veryrapid and accurate detection of nucleic acids are therefore needed fortimely diagnosis and treatment of disease, to identify toxins inconsumables and the environment, as well as other applications. Inaddition, being able to “tune” an assay to detect target nucleic acidsinstantaneously or over a longer period of time, and to be able toquantify the targets very accurately provides flexibility for virtuallyany application.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description, including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure provides compositions of matter and assay methodsto detect target nucleic acids of interest where reaction kinetics ofthe assay can be controlled via molecular design of one of the reactioncomponents. The “nucleic acid-guided nuclease cascade assays” or “signalboost cascade assays” or “cascade assays” described herein comprise twodifferent ribonucleoprotein complexes and either blocked nucleic acidmolecules or blocked primer molecules. The blocked nucleic acidmolecules or blocked primer molecules keep one of the ribonucleoproteincomplexes “locked” unless and until a target nucleic acid of interestactivates the other ribonucleoprotein complex. In the context of thecascade assays, “locked” means that the blocked nucleic acid moleculesor blocked primer molecules are designed in such a way that they arelargely blocked from interacting with second ribonucleoproteincomplexes; therefore, the ribonucleoprotein complexes remain largelyinactive (i.e., “locked”) unless and until a target nucleic acid ofinterest activates the first ribonucleoprotein complex. The presentnucleic acid-guided nuclease cascade assay can detect one or more targetnucleic acids of interest (e.g., DNA, RNA and/or cDNA) at attamolar (aM)(or lower) limits in some embodiments virtually instantaneously withoutthe need for amplifying the target nucleic acid(s) of interest, therebyavoiding the drawbacks of multiplex amplification, such asprimer-dimerization. Further, the cascade assay allows for “tuning” ofassay kinetics to alter detection times of target nucleic acids ofinterest from instantaneous to over 100 minutes or more, and overvarying concentration ranges of the target nucleic acid of interest from1 copy to 10,000 copies and/or discerning 1 copy of the target nucleicacids of interest from 2 copies of the target nucleic acid of interest.A particularly advantageous feature of the cascade assay generally isthat, with the exception of the guide nucleic acid in RNP1, the cascadeassay components can be the same in each assay no matter what targetnucleic acid(s) of interest is being detected.

A first exemplary embodiment of the disclosure provides a tunableblocked nucleic acid molecule comprising: a first region recognized by aribonucleoprotein (RNP) complex; one or more second regions notcomplementary to the first region forming at least one loop; and one ormore third regions complementary to and hybridized to the first regionforming at least one clamp, wherein the Gibbs free energy of the tunableblocked nucleic acid molecule at 25° C. is at most about −5 kcal/molwhen the following formula is used to calculate the free energy for eachbase pair: ΔG° (T)=(ΔH°−TΔS°)cal mol⁻¹, and total ΔG° is given by: ΔG°(total)=Σ_(i)n_(i) ΔG° (i)+ΔG° (init with term G·C)+ΔG° (init with termA·T)+ΔG° (sym), where ΔG° (i) are the standard free energy changes forthe 10 possible Watson-Crick NNs (e.g., ΔG° (1)=ΔG°₃₇ (AA/TT), ΔG°(2)=ΔG°₃₇ (TA/AT), . . . etc.), n; is the number of occurrences of eachnearest neighbor, i, and ΔG° (sym) equals+0.43 kcal/mol (1 cal=4.184 J)if the duplex is self-complementary and zero if it isnon-self-complementary, and wherein cleavage of the one or more secondregions results in dehybridization of the one or more the third regionsfrom the first region, resulting in an unblocked nucleic acid molecule.

In some aspects, the tunable blocked nucleic acid molecule comprises astructure represented by any one of Formulas I-N, wherein Formulas I-Nare in the 5′-to-3′ direction:(a) A-(B-L)_(J)-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;    -   B is 4-12 nucleotides in length;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10;    -   C is 4-15 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then A-(B-L)_(J)-C and T-D are separate nucleic acid        strands;    -   T is 17-135 nucleotides in length and comprises at least 50%        sequence complementarity to B and C; and    -   D is 0-10 nucleotides in length and comprises at least 50%        sequence complementarity to A;        (b) D-T-T′-C-(L-B)_(J)-A  (Formula II);    -   wherein D is 0-10 nucleotides in length;    -   T-T′ is 17-135 nucleotides in length;    -   T is 1-10 nucleotides in length and does not hybridize with T;    -   C is 4-15 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length and does not hybridize with T;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   J is an integer between 1 and 10;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;        (c) T-D-M-A-(B-L)_(J)-C  (Formula III);    -   wherein T is 17-135 nucleotides in length;    -   D is 0-10 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then T-D and A-(B-L)_(J)-C are separate nucleic acid        strands;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10; and    -   C is 4-15 nucleotides in length; or        (d) T-D-M-A-L_(P)-C  (Formula N);    -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-15 nucleotides in length;    -   M is 1-25 nucleotides in length;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D; and    -   L is 3-25 nucleotides in length;    -   p is 0or 1;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T.        And in some aspects, in the tunable blocked nucleic acid        molecule:    -   (a) T of Formula I comprises at least 80% sequence        complementarity to B and C;    -   (b) D of Formula I comprises at least 80% sequence        complementarity to A;    -   (c) C of Formula II comprises at least 80% sequence        complementarity to T;    -   (d) B of Formula II comprises at least 80% sequence        complementarity to T;    -   (e) A of Formula II comprises at least 80% sequence        complementarity to D;    -   (f) A of Formula III comprises at least 80% sequence        complementarity to D;    -   (g) B of Formula III comprises at least 80% sequence        complementarity to T;    -   (h) A of Formula IV comprises at least 80% sequence        complementarity to D; and/or    -   (i) C of Formula N comprises at least 80% sequence        complementarity to T.

In some aspects, the tunable blocked nucleic acid molecule comprises amodified nucleoside or nucleotide, and in some aspects, the modifiednucleoside or nucleotide comprises a locked nucleic acid (LNA), apeptide nucleic acid (PNA), a 2′-O-methyl (2′-O-Me) modified nucleoside,a 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS)bond.

In some aspects of the first exemplary embodiment, the tunable blockednucleic acid molecule is a tunable blocked primer molecule.

In some aspects, the tunable blocked nucleic acid molecule does notcomprise a PAM sequence, and in some aspects, the tunable blockednucleic acid molecule comprises a PAM sequence in the one or more secondregions not complementary to the first region forming at least one loop.

In some aspects, the tunable blocked nucleic acid molecule furthercomprises a reporter moiety, wherein the reporter moiety comprises aDNA, RNA or chimeric nucleic acid molecule, is operably linked to thetunable blocked nucleic acid molecule and produces a detectable signalupon cleavage by the ribonucleoprotein complex. In some aspects, thedetectable signal is a fluorescent, chemiluminescent, radioactive,colorimetric or other optical signal.

In some aspects there is provided a ribonucleoprotein complex comprisingthe tunable blocked nucleic acid molecule that has become unblocked.

In some aspects, the tunable blocked nucleic acid molecule has a freeenergy at 25° C. of at most about −5.5 kcal/mol and detection of thetarget nucleic acid of interest occurs instantaneously. In some aspects,the tunable blocked nucleic acid molecule has a free energy at 25° C. ofat most about −7.0 kcal/mol, or at most about −8.0 kcal/mol, or at mostabout −10.0 kcal/mol, or at most about −12.0 kcal/mol, or at most about−13.0 kcal/mol, or at most about −15.0 kcal/mol, or at most about −17.5kcal/mol, or at most about −19.0 kcal/mol, or at most about −20.0kcal/mol. In some aspects, the free energy of the tunable blockednucleic acid molecule at 25° C. is at most about −5.5 kcal/mol to about−20.0 kcal/mol. In some aspects, the free energy of the tunable blockednucleic acid molecule at 25° C. is at most about −10.0 kcal/mol −20.0kcal/mol.

In some aspects, the tunable blocked nucleic acid molecule comprises atleast 2 second regions; in some aspects, the tunable blocked nucleicacid molecule comprises at least 3 second regions, and in some aspects,the tunable blocked nucleic acid molecule comprises at least 4 secondregions.

In some aspects of the first exemplary embodiment, the tunable blockednucleic acid molecule comprises two separate but complementaryoligonucleotides.

In an exemplary second embodiment of the disclosure there is provided areaction mixture comprising: a first ribonucleoprotein (RNP) complex(RNP1) comprising a first nucleic acid-guided nuclease and a first guideRNA (gRNA); wherein the first gRNA comprises a sequence complementary toa target nucleic acid of interest, and wherein the first nucleicacid-guided nuclease exhibits both cis-cleavage activity andtrans-cleavage activity; a second ribonucleoprotein complex (RNP2)comprising a second nucleic acid-guided nuclease and a second gRNA thatis not complementary to the target nucleic acid of interest; wherein thesecond nucleic acid-guided nuclease exhibits both cis-cleavage activityand trans-cleavage activity; and a plurality of the tunable blockednucleic acid molecules, wherein the tunable blocked nucleic acidcomprises a first region recognized by the RNP2; one or more secondregions not complementary to the first region forming at least one loop;and one or more third regions complementary to and hybridized to thefirst region forming at least one clamp, wherein the free energy of thetunable blocked nucleic acid molecule at 25° C. is at most about −5kcal/mol when the following formula is used to calculate the free energyfor each base pair: ΔG° (T)=(ΔH°−TΔS°)cal mol⁻¹, and total ΔG° is givenby: ΔG° (total)=Σ_(i)n_(i) ΔG° (i)+ΔG° (init with term G·C)+ΔG° (initwith term A·T)+ΔG° (sym), where ΔG° (i) are the standard free energychanges for the 10 possible Watson-Crick NNs (e.g., ΔG° (1)=ΔG°₃₇(AA/TT), ΔG° (2)=ΔG°₃₇ (TA/AT), . . . etc.), n; is the number ofoccurrences of each nearest neighbor, i, and ΔG° (sym) equals+0.43kcal/mol (1 cal=4.184 J) if the duplex is self-complementary and zero ifit is non-self-complementary, and wherein cleavage of the one or moresecond regions results in dehybridization of the one or more the thirdregions from the first region, resulting in an unblocked nucleic acidmolecule.

In an exemplary third embodiment of the disclosure there is provided areaction mixture comprising: a first ribonucleoprotein (RNP) complex(RNP1) comprising a first nucleic acid-guided nuclease and a first guideRNA (gRNA); wherein the first gRNA comprises a sequence complementary toa target nucleic acid of interest, and wherein the first nucleicacid-guided nuclease exhibits both cis-cleavage activity andtrans-cleavage activity; a second ribonucleoprotein complex (RNP2)comprising a second nucleic acid-guided nuclease and a second gRNA thatis not complementary to the target nucleic acid of interest; wherein thesecond nucleic acid-guided nuclease exhibits both cis-cleavage activityand trans-cleavage activity; a plurality of template moleculescomprising a sequence corresponding to the second gRNA; a plurality oftunable blocked primer molecules comprising a sequence complementary tothe template molecules, wherein the tunable blocked primer moleculescannot be extended by a polymerase and wherein the tunable blockedprimer molecule comprises a first region recognized by the RNP2; one ormore second regions not complementary to the first region forming atleast one loop; and one or more third regions complementary to andhybridized to the first region forming at least one clamp, wherein thefree energy of the tunable blocked nucleic acid molecule at 25° C. is atmost about −5 kcal/mol when the following formula is used to calculatethe free energy for each base pair: ΔG° (T)=(ΔH°−TΔS°)cal mol⁻¹, andtotal ΔG° is given by: ΔG°(total)=Σ_(i)n_(i) ΔG° (i)+ΔG° (init with termG·C)+ΔG° (init with term A·T)+ΔG° (sym), where ΔG° (i) are the standardfree energy changes for the 10 possible Watson-Crick NNs (e.g., ΔG°(1)=ΔG°₃₇ (AA/TT), ΔG° (2)=ΔG°₃₇ (TA/AT), . . . etc.), n; is the numberof occurrences of each nearest neighbor, i, and ΔG° (sym) equals +0.43kcal/mol (1 cal=4.184 J) if the duplex is self-complementary and zero ifit is non-self-complementary, and wherein cleavage of the one or moresecond regions results in dehybridization of the one or more the thirdregions from the first region, resulting in an unblocked nucleic acidmolecule; and a polymerase and a plurality of nucleotides.

In some aspects of the exemplary second and third embodiments, one orboth of the RNP1 and the RNP2 comprise a nucleic acid-guided nucleaseselected from Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14,Cas12h, Cas12i, Cas12j, Cas13a, or Cas13b; and in some aspects, one orboth of the RNP1 or the RNP2 comprise a nucleic acid-guided nucleasethat is a Type V nucleic acid-guided nuclease or a Type VI nucleicacid-guided nuclease; and in some aspects, one or both of RNP1 and RNP2comprises a nucleic acid-guided nuclease comprising a RuvC nucleasedomain or a RuvC-like nuclease domain but lacks an HNH nuclease domain.

In some aspects, the tunable blocked nucleic acid molecule or tunableblocked primer molecule of the reaction mixture further comprises areporter moiety, and wherein upon binding of a target nucleic acid ofinterest to RNP1, a signal from the reporter moiety is detected.

In some aspects of the exemplary second and third embodiments, thetunable blocked nucleic acid molecule or tunable blocked primer moleculeof the reaction mixture comprises a structure represented by any one ofFormulas I-N, wherein Formulas I-N are in the 5′-to-3′ direction:(a) A-(B-L)_(J)-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;    -   B is 4-12 nucleotides in length;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10;    -   C is 4-15 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then A-(B-L)J-C and T-D are separate nucleic acid        strands;    -   T is 17-135 nucleotides in length and comprises at least 50%        sequence complementarity to B and C; and    -   D is 0-10 nucleotides in length and comprises at least 50%        sequence complementarity to A;        (b) D-T-T′-C-(L-B)_(J)-A  (Formula II);    -   wherein D is 0-10 nucleotides in length;    -   T-T′ is 17-135 nucleotides in length;    -   T is 1-10 nucleotides in length and does not hybridize with T;    -   C is 4-15 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length and does not hybridize with T;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   J is an integer between 1 and 10;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;        (c) T-D-M-A-(B-L)_(J)-C  (Formula III);    -   wherein T is 17-135 nucleotides in length;    -   D is 0-10 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then T-D and A-(B-L)_(J)-C are separate nucleic acid        strands;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10; and    -   C is 4-15 nucleotides in length; or        (d) T-D-M-A-L_(P)-C  (Formula N);    -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-15 nucleotides in length;    -   M is 1-25 nucleotides in length;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D; and    -   L is 3-25 nucleotides in length;    -   p is 0or 1;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T.

In some aspects, the tunable blocked nucleic acid molecule or tunableblocked primer molecule of the reaction mixture has a free energy at 25°C. of at most about −5.5 kcal/mol and detection of the target nucleicacid of interest occurs instantaneously. In some aspects, the tunableblocked nucleic acid molecule or tunable blocked primer molecule has afree energy at 25° C. of at most about −7.0 kcal/mol, or at most about−8.0 kcal/mol, or at most about −10.0 kcal/mol, or at most about −12.0kcal/mol, or at most about −13.0 kcal/mol, or at most about −15.0kcal/mol, or at most about −17.5 kcal/mol, or at most about −19.0kcal/mol, or at most about −20.0 kcal/mol. In some aspects, the freeenergy of the tunable blocked nucleic acid molecule or tunable blockedprimer molecule at 25° C. is at most about −5.5 kcal/mol to about −20.0kcal/mol. In some aspects, the free energy of the tunable blockednucleic acid molecule or tunable blocked primer molecule at 25° C. is atmost about −10.0 kcal/mol −20.0 kcal/mol.

In some aspects, the tunable blocked nucleic acid molecule or tunableblocked primer molecule of the reaction mixture comprises at least 2second regions; in some aspects, the tunable blocked nucleic acidmolecule or tunable blocked primer molecule comprises at least 3 secondregions, and in some aspects, the tunable blocked nucleic acid moleculeor tunable blocked primer molecule comprises at least 4 second regions.

In some aspects of the exemplary second and third embodiments, thetunable blocked primer molecule comprises two separate but complementaryoligonucleotides.

In some aspects of the reaction mixtures, the tunable blocked nucleicacid molecule or tunable blocked primer molecule comprises a singlepartially self-hybridizing oligonucleotide.

In some aspects, the tunable blocked nucleic acid molecule or tunableblocked primer molecule of the reaction mixture comprises a modifiednucleoside or nucleotide, and in some aspects, the modified nucleosideor nucleotide comprises a locked nucleic acid (LNA), a peptide nucleicacid (PNA), a 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro(2′-F) modified nucleoside, and/or a phosphorothioate (PS) bond.

A fourth exemplary embodiment of the disclosure provides a method fordetecting a nucleic acid target of interest in a sample comprising thesteps of: providing reaction mix comprising: first ribonucleoproteincomplexes (RNP1 s), wherein the RNP1 s comprise a first nucleicacid-guided nuclease and a first gRNA; wherein the first gRNA comprisesa sequence complementary to the nucleic acid target of interest, andwherein the first nucleic acid-guided nuclease exhibits bothcis-cleavage activity and trans-cleavage activity; secondribonucleoprotein complexes (RNP2s), wherein the RNP2s comprise a secondnucleic acid-guided nuclease and a second gRNA that is not complementaryto the aptamer complement, and wherein the second nucleic acid-guidednuclease exhibits both cis- and trans-cleavage activity; and a pluralityof tunable blocked nucleic acid molecules, wherein the tunable blockednucleic acid molecules comprise: a first region recognized by the RNP2complex; one or more second regions not complementary to the firstregion forming at least one loop; and one or more third regionscomplementary to and hybridized to the first region forming at least oneclamp, wherein the free energy of the plurality of tunable blockedprimer molecules at 25° C. are at most about −5 kcal/mol when thefollowing formula is used to calculate the free energy for each basepair: ΔG° (T)=(ΔH°−TΔS°)cal mol⁻¹, and total ΔG° is given by: ΔG°(total)=Σ_(i)n_(i) ΔG° (i)+ΔG° (init with term G·C)+ΔG° (init with termA·T)+ΔG° (sym), where ΔG° (i) are the standard free energy changes forthe 10 possible Watson-Crick NNs (e.g., ΔG° (1)=ΔG°₃₇ (AA/TT), ΔG°(2)=ΔG°₃₇ (TA/AT), . . . etc.), n; is the number of occurrences of eachnearest neighbor, i, and ΔG° (sym) equals +0.43 kcal/mol (1 cal=4.184 J)if the duplex is self-complementary and zero if it isnon-self-complementary, and wherein cleavage of the one or more secondregions results in dehybridization of the one or more the third regionsfrom the first region, resulting in an unblocked nucleic acid molecule;contacting the reaction mixture with the sample under conditions thatallow non-nucleic acid targets of interest in the sample to bind to theRNP1, wherein: upon binding of the target nucleic acid of interest tothe RNP1, the RNP1 becomes active trans-cleaving at least one of thetunable blocked nucleic acid molecules, thereby producing at least oneunblocked nucleic acid molecule that can complex with the RNP2; and uponbinding of the at least one unblocked nucleic acid molecule to the RNP2,the RNP2 becomes active trans-cleaving at least one more of the tunableblocked nucleic acid molecules; allowing the cascade to continue; anddetecting the unblocked nucleic acid molecules, thereby detecting thetarget nucleic acid of interest in the sample.

A fifth exemplary embodiment of the disclosure provides a method fordetecting a nucleic acid target of interest in a sample comprising thesteps of: providing reaction mix comprising: first ribonucleoproteincomplexes (RNP1 s), wherein the RNP1 s comprise a first nucleicacid-guided nuclease and a first guide RNA (gRNA); wherein the firstgRNA comprises a sequence complementary to the nucleic acid target ofinterest, and wherein the first nucleic acid-guided nuclease exhibitsboth cis-cleavage activity and trans-cleavage activity; secondribonucleoprotein complexes (RNP2s) comprising a second nucleicacid-guided nuclease and a second gRNA that is not complementary to thetarget nucleic acid of interest; wherein the second nucleic acid-guidednuclease exhibits both cis-cleavage activity and trans-cleavageactivity; a plurality of template molecules comprising sequence homologyto the second gRNA; a plurality of tunable blocked primer moleculescomprising a sequence complementary to the template molecules, whereinthe tunable blocked primer molecules cannot be extended by a polymerase,and wherein the tunable blocked primer molecules comprise: a firstregion recognized by the RNP2; one or more second regions notcomplementary to the first region forming at least one loop; and one ormore third regions complementary to and hybridized to the first regionforming at least one clamp, wherein the free energy of the tunableblocked primer molecules at 25° C. are at most about −5 kcal/mol whenthe following formula is used to calculate the free energy for each basepair: ΔG° (T)=(ΔH°−TΔS°)cal mol⁻¹, and total ΔG° is given by: ΔG°(total)=Σ_(i)n_(i) ΔG°(i)+ΔG° (init with term G·C)+ΔG° (init with termA·T)+ΔG° (sym), where ΔG° (i) are the standard free energy changes forthe 10 possible Watson-Crick NNs (e.g., ΔG° (1)=ΔG°₃₇ (AA/TT), ΔG°(2)=ΔG°₃₇ (TA/AT), . . . etc.), n; is the number of occurrences of eachnearest neighbor, i, and ΔG° (sym) equals +0.43 kcal/mol (1 cal=4.184 J)if the duplex is self-complementary and zero if it isnon-self-complementary, and wherein cleavage of the one or more secondregions results in dehybridization of the one or more the third regionsfrom the first region, resulting in an unblocked nucleic acid molecule;and a polymerase and a plurality of nucleotides; contacting the reactionmixture with the sample under conditions that allow nucleic acid targetsof interest in the sample to bind to RNP1, wherein: upon binding of thenucleic acid targets of interest to the RNP1, the RNP1 becomes activetrans-cleaving at least one of the tunable blocked primer molecules,thereby producing at least one unblocked primer molecule that can beextended by the polymerase; the at least one unblocked primer moleculebinds to one of the template molecules and is extended by the polymeraseand nucleotides to form at least one synthesized activating moleculehaving a sequence complementary to the second gRNA; and the at least onesynthesized activating molecule binds to the second gRNA, and RNP2becomes active cleaving at least one further tunable blocked primermolecule and at least one reporter moiety in a cascade; allowing thecascade to continue; and detecting the unblocked primer molecules,thereby detecting the target nucleic acid of interest in the sample.

In some aspects of the exemplary fourth and fifth embodiments, thereaction mix further comprises reporter moieties, wherein the reportermoieties produce a detectable signal upon trans-cleavage activity by theRNP2 to identify the presence of one or more nucleic acid targets ofinterest in the sample. In some aspects, the tunable blocked nucleicacid molecule further comprises the reporter moiety, and wherein upondetection of a target nucleic acid of interest, a signal from thereporter moiety is detected.

Also in some embodiments of the exemplary fourth and fifth embodiments,one or both of the RNP1 and the RNP2 comprise a nucleic acid-guidednuclease selected from Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e,Cas14, Cas12h, Cas12i, Cas12j, Cas13a, or Cas13b; and in some aspects,one or both of the RNP1 and the RNP2 comprise a nucleic acid-guidednuclease that is a Type V nucleic acid-guided nuclease or a Type VInucleic acid-guided nuclease.

In some aspects of the exemplary fourth and fifth embodiments, thetunable blocked nucleic acid molecule or tunable blocked primer moleculecomprises a structure represented by any one of Formulas I-N, whereinFormulas I-N are in the 5′-to-3′ direction:(e) A-(B-L)_(J)-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;    -   B is 4-12 nucleotides in length;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10;    -   C is 4-15 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then A-(B-L)J-C and T-D are separate nucleic acid        strands;    -   T is 17-135 nucleotides in length and comprises at least 50%        sequence complementarity to B and C; and    -   D is 0-10 nucleotides in length and comprises at least 50%        sequence complementarity to A;        (f) D-T-T′-C-(L-B)_(J)-A  (Formula II);    -   wherein D is 0-10 nucleotides in length;    -   T-T′ is 17-135 nucleotides in length;    -   T is 1-10 nucleotides in length and does not hybridize with T;    -   C is 4-15 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length and does not hybridize with T;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   J is an integer between 1 and 10;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;        (g) T-D-M-A-(B-L)_(J)-C  (Formula III);    -   wherein T is 17-135 nucleotides in length;    -   D is 0-10 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then T-D and A-(B-L)_(J)-C are separate nucleic acid        strands;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10; and    -   C is 4-15 nucleotides in length; or        (h) T-D-M-A-L_(P)-C  (Formula N);    -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-15 nucleotides in length;    -   M is 1-25 nucleotides in length;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D; and    -   L is 3-25 nucleotides in length;    -   p is 0 or 1;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T.

In some aspects, the tunable blocked nucleic acid molecule or tunableblocked primer molecule of the reaction mixture has a free energy at 25°C. of at most about −5.5 kcal/mol and detection of the target nucleicacid of interest occurs instantaneously. In some aspects, the tunableblocked nucleic acid molecule or tunable blocked primer molecule has afree energy at 25° C. of at most about −7.0 kcal/mol, or at most about−8.0 kcal/mol, or at most about −10.0 kcal/mol, or at most about −12.0kcal/mol, or at most about −13.0 kcal/mol, or at most about −15.0kcal/mol, or at most about −17.5 kcal/mol, or at most about −19.0kcal/mol, or at most about −20.0 kcal/mol. In some aspects, the freeenergy of the tunable blocked nucleic acid molecule or tunable blockedprimer molecule at 25° C. is at most about −5.5 kcal/mol to about −20.0kcal/mol. In some aspects, the free energy of the tunable blockednucleic acid molecule or tunable blocked primer molecule at 25° C. is atmost about −10.0 kcal/mol −20.0 kcal/mol.

In some aspects, the tunable blocked nucleic acid molecule or tunableblocked primer molecule of the reaction mixture comprises at least 2second regions; in some aspects, the tunable blocked nucleic acidmolecule or tunable blocked primer molecule comprises at least 3 secondregions, and in some aspects, the tunable blocked nucleic acid moleculeor tunable blocked primer molecule comprises at least 4 second regions.

In some aspects of the exemplary fourth and fifth embodiments, thetunable blocked nucleic acid molecule or tunable blocked primer moleculecomprises two separate but complementary oligonucleotides, yet in otheraspects, the tunable blocked nucleic acid molecule or tunable blockedprimer molecule comprises a single partially self-hybridizingoligonucleotide.

In some aspects of the methods, the tunable blocked nucleic acidmolecule or tunable blocked primer molecule comprises a modifiednucleoside or nucleotide, and in some aspects, the modified nucleosideor nucleotide comprises a locked nucleic acid (LNA), a peptide nucleicacid (PNA), a 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro(2′-F) modified nucleoside, and/or a phosphorothioate (PS) bond.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1A is an overview of a prior art quantitative PCR (“qPCR”) assaywhere target nucleic acids of interest from a sample are amplifiedbefore detection.

FIG. 1B is an overview of a CRISPR-based prior art assay where targetnucleic acids of interest from a sample must be amplified beforeperforming the detection assay.

FIG. 1C is an overview of the general principles underlying the nucleicacid-guided nuclease cascade assay described in detail herein wheretarget nucleic acids of interest from a sample do not need to beamplified before detection.

FIG. 2A is a diagram showing the sequence of steps in an exemplarycascade assay utilizing blocked nucleic acids.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid moleculeand a method for unblocking the blocked nucleic acid molecules of thedisclosure.

FIG. 2C shows schematics of several exemplary blocked nucleic acidmolecules containing the structure of Formula I, as described herein.

FIG. 2D shows schematics of several exemplary blocked nucleic acidmolecules containing the structure of Formula II, as described herein.

FIG. 2E shows schematics of several exemplary blocked nucleic acidmolecules containing the structure of Formula III, as described herein.

FIG. 2F shows schematics of two exemplary blocked nucleic acid moleculescontaining the structure of Formula IV, as described herein.

FIG. 3A is a diagram showing the sequence of steps in an exemplarycascade assay involving circular blocked primer molecules and lineartemplate molecules.

FIG. 3B is a diagram showing the sequence of steps in an exemplarycascade assay involving circular blocked primer molecules and circulartemplate molecules.

FIG. 4 illustrates three embodiments of reporter moieties.

FIG. 5 is an illustration of a lateral flow assay that can be used todetect the cleavage and separation of a signal from a reporter moiety.

FIG. 6A depicts Molecule U29 and describes the properties thereof, whereU29 was used to generate the data shown in FIGS. 6B-6H.

FIG. 7A depicts Molecule F375 and describes the properties thereof,where Molecule F375 was used to generate the data shown in FIG. 7B.

FIG. 8A depicts Molecule U250 and describes the properties thereof,where Molecule U250 was used to generate the data shown in FIG. 8B.

FIG. 9A depicts Molecule T135 and describes the properties thereof,where Molecule T135 was used to generate the data shown in FIG. 9B.

FIG. 10A depicts Molecule T134 and describes the properties thereof,where Molecule T134 was used to generate the data shown in FIG. 10B.

FIG. 11A depicts Molecule T119 and describes the properties thereof,where Molecule T119 was used to generate the data shown in FIG. 11B.

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

Definitions

All of the functionalities described in connection with one embodimentof the compositions and/or methods described herein are intended to beapplicable to the additional embodiments of the compositions and/ormethods except where expressly stated or where the feature or functionis incompatible with the additional embodiments. For example, where agiven feature or function is expressly described in connection with oneembodiment but not expressly mentioned in connection with an alternativeembodiment, it should be understood that the feature or function may bedeployed, utilized, or implemented in connection with the alternativeembodiment unless the feature or function is incompatible with thealternative embodiment.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “a system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth. Additionally, it is to be understood that terms such as“left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”“outer” that may be used herein merely describe points of reference anddo not necessarily limit embodiments of the present disclosure to anyparticular orientation or configuration. Furthermore, terms such as“first,” “second,” “third,” etc., merely identify one of a number ofportions, components, steps, operations, functions, and/or points ofreference as disclosed herein, and likewise do not necessarily limitembodiments of the present disclosure to any particular configuration ororientation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention. Conventional methodsare used for the procedures described herein, such as those provided inthe art, and demonstrated in the Examples and various generalreferences. Unless otherwise stated, nucleic acid sequences describedherein are given, when read from left to right, in the 5′ to 3′direction. Nucleic acid sequences may be provided as DNA, as RNA, or acombination of DNA and RNA (e.g., a chimeric nucleic acid).

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in smaller ranges, and arealso encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the multiple specified features or components withor without another. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention. The terms used herein are intended to have the plain andordinary meaning as understood by those of ordinary skill in the art.

As used herein, the term “about,” as applied to one or more values ofinterest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1%, or less in either direction (greater than or less than)of a stated reference value, unless otherwise stated or otherwiseevident from the context (except where such number would exceed 100% ofa possible value).

As used herein, the terms “binding affinity” or “dissociation constant”or “K_(d)” refer to the tendency of a molecule to bind (covalently ornon-covalently) to a different molecule. A high K_(d) (which in thecontext of the present disclosure refers to blocked nucleic acidmolecules or blocked primer molecules binding to RNP2) indicates thepresence of more unbound molecules, and a low IQ (which in the contextof the present disclosure refers to unblocked nucleic acid molecules orunblocked primer molecules binding to RNP2) indicates the presence ofmore bound molecules. In the context of the present disclosure and thebinding of blocked or unblocked nucleic acid molecules or blocked orunblocked primer molecules to RNP2, low IQ values are in a range fromabout 100 fM to about 1 aM or lower (e.g., 100 zM) and high K_(d) valuesare in the range of 100 nM-100 μM (10 mM) and thus are about 10⁵-to10¹⁰-fold or higher as compared to low K_(d) values.

As used herein, the terms “binding domain” or “binding site” refer to aregion on a protein, DNA, or RNA, to which specific molecules and/orions (ligands) may form a covalent or non-covalent bond. By way ofexample, a polynucleotide sequence present on a nucleic acid molecule(e.g., a primer binding domain) may serve as a binding domain for adifferent nucleic acid molecule (e.g., an unblocked primer nucleic acidmolecule). Characteristics of binding sites are chemical specificity, ameasure of the types of ligands that will bond, and affinity, which is ameasure of the strength of the chemical bond.

As used herein, the term “blocked nucleic acid molecule” refers tonucleic acid molecules that cannot bind to the first or second RNPcomplex (i.e., RNP1 or RNP2) to activate cis- or trans-cleavage.“Unblocked nucleic acid molecule” refers to a formerly blocked nucleicacid molecule that can bind to the second RNP complex (RNP2) to activatetrans-cleavage of additional blocked nucleic acid molecules. A “blockednucleic acid molecule” may be a “blocked primer molecule” in someembodiments of the cascade assay.

The terms “Cos RNA-guided endonuclease” or “CRISPR nuclease” or “nucleicacid-guided nuclease” refer to a CRISPR-associated protein that is anRNA-guided endonuclease suitable for assembly with a sequence-specificgRNA to form a ribonucleoprotein (RNP) complex.

As used herein, the terms “cis-cleavage”, “cis-endonuclease activity”,“cis-mediated endonuclease activity”, “cis-nuclease activity”,“cis-mediated nuclease activity”, and variations thereof refer tosequence-specific cleavage of a target nucleic acid of interest,including an unblocked nucleic acid molecule or synthesized activatingmolecule, by a nucleic acid-guided nuclease in an RNP complex.Cis-cleavage is a single turn-over cleavage event in that only onesubstrate molecule is cleaved per event.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen-bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” or“percent homology” to a specified second nucleotide sequence. Forexample, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10, or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3; and the nucleotide sequence 3′-ATCGAT-5′is 100% complementary to a region of the nucleotide sequence5′-GCTAGCTAG-3′.

As used herein, the term “contacting” refers to placement of twomoieties in direct physical association, including in solid or liquidform. Contacting can occur in vitro with isolated cells (for example ina tissue culture dish or other vessel) or in samples or in vivo byadministering an agent to a subject.

A “control” is a reference standard of a known value or range of values.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a crRNA region or guide sequence capable ofhybridizing to the target strand of a target nucleic acid of interest,and 2) a scaffold sequence capable of interacting or complexing with anucleic acid-guided nuclease. The crRNA region of the gRNA is acustomizable component that enables specificity in every nucleicacid-guided nuclease reaction. A gRNA can include any polynucleotidesequence having sufficient complementarity with a target nucleic acid ofinterest to hybridize with the target nucleic acid of interest and todirect sequence-specific binding of a ribonucleoprotein (RNP) complexcontaining the gRNA and nucleic acid-guided nuclease to the targetnucleic acid. Target nucleic acids of interest may include a protospaceradjacent motif (PAM), and, following gRNA binding, the nucleicacid-guided nuclease induces a double-stranded break either inside oroutside the protospacer region on the target nucleic acid of interest,including on an unblocked nucleic acid molecule or synthesizedactivating molecule. A gRNA may contain a spacer sequence including aplurality of bases complementary to a protospacer sequence in the targetnucleic acid. For example, a spacer can contain about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, ormore bases. The gRNA spacer may be 50%, 60%, 75%, 80%, 85%, 90%, 95%,97.5%, 98%, 99%, or more complementary to its corresponding targetnucleic acid of interest. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences. A guide RNA may befrom about 20 nucleotides to about 300 nucleotides long. Guide RNAs maybe produced synthetically or generated from a DNA template.

“Modified” refers to a changed state or structure of a molecule.Molecules may be modified in many ways including chemically,structurally, and functionally. In one embodiment, a nucleic acidmolecule (for example, a blocked nucleic acid molecule) may be modifiedby the introduction of non-natural nucleosides, nucleotides, and/orinternucleoside linkages. In another embodiment, a modified protein(e.g., a nucleic acid-guided nuclease) may refer to any polypeptidesequence alteration which is different from the wildtype.

The terms “percent sequence identity”, “percent identity”, or “sequenceidentity” refer to percent (%) sequence identity with respect to areference polynucleotide or polypeptide sequence following alignment bystandard techniques. Alignment for purposes of determining percentsequence identity can be achieved in various ways that are within thecapabilities of one of skill in the art, for example, using publiclyavailable computer software such as BLAST, BLAST-2, PSI-BLAST, orMEGALIGN™ software. In some embodiments, the software is MUSCLE (Edgar,Nucleic Acids Res., 32(5):1792 1797 (2004)). Those skilled in the artcan determine appropriate parameters for aligning sequences, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For example, in embodiments, percentsequence identity values are generated using the sequence comparisoncomputer program BLAST (Altschul, et al., J. Mol. Biol., 215:403-410(1990)).

As used herein, the terms “preassembled ribonucleoprotein complex”,“ribonucleoprotein complex”, “RNP complex”, or “RNP” refer to a complexcontaining a guide RNA (gRNA) and a nucleic acid-guided nuclease, wherethe gRNA is integrated with the nucleic acid-guided nuclease. The gRNA,which includes a sequence complementary to a target nucleic acid ofinterest, guides the RNP to the target nucleic acid of interest andhybridizes to it. The hybridized target nucleic acid-gRNA units arecleaved by the nucleic acid-guided nuclease. In the cascade assaysdescribed herein, a first ribonucleoprotein complex (RNP1) includes afirst guide RNA (gRNA) specific to a nucleic acid target nucleic acid ofinterest, and a first nucleic acid-guided nuclease, such as, forexample, cas 12a or cas 14a for a DNA target nucleic acid, or cas 13afor an RNA target nucleic acid. A second ribonucleoprotein complex(RNP2) for signal amplification includes a second guide RNA specific toan unblocked nucleic acid or synthesized activating molecule, and asecond nucleic acid-guided nuclease, which may be different from or thesame as the first nucleic acid-guided nuclease.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably. Proteins may or may not be made up entirely of aminoacids.

As used herein, the term “sample” refers to tissues; cells or componentparts; body fluids, including but not limited to peripheral blood,serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum,saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid,cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostaticfluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter,hair, tears, cyst fluid, pleural and peritoneal fluid, pericardialfluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus,sebum, vomit, vaginal secretions, mucosal secretion, stool water,pancreatic juice, lavage fluids from sinus cavities, bronchopulmonaryaspirates, blastocyl cavity fluid, and umbilical cord blood. “Sample”may also refer to specimen or aliquots from food; agricultural products;pharmaceuticals; cosmetics, nutraceuticals; personal care products;environmental substances such as soil, water, air, or sewer sample;industrial sites and products; and chemicals and compounds. A samplefurther may include a homogenate, lysate or extract. A sample furtherrefers to a medium, such as a nutrient broth or gel, which may containcellular components, such as proteins or nucleic acid molecules.

The terms “target DNA sequence”, “target sequence”, “target nucleic acidof interest”, “target molecule of interest”, “target nucleic acid”, or“target of interest” refer to any locus that is recognized by a gRNAsequence in vitro or in vivo. The “target strand” of a target nucleicacid of interest is the strand of the double-stranded target nucleicacid that is complementary to a gRNA. The spacer sequence of a gRNA maybe 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99% or morecomplementary to the target nucleic acid of interest. Optimal alignmentcan be determined with the use of any suitable algorithm for aligningsequences. Full complementarity is not necessarily required providedthere is sufficient complementarity to cause hybridization andtrans-cleavage activation of an RNP complex. A target nucleic acid ofinterest can include any polynucleotide, such as DNA (ssDNA or dsDNA) orRNA polynucleotides. A target nucleic acid of interest may be located inthe nucleus or cytoplasm of a cell such as, for example, within anorganelle of a eukaryotic cell, such as a mitochondrion or achloroplast, or it can be exogenous to a host cell, such as a eukaryoticcell or a prokaryotic cell. The target nucleic acid of interest may bepresent in a sample, such as a biological or environmental sample, andit can be a viral nucleic acid molecule, a bacterial nucleic acidmolecule, a fungal nucleic acid molecule, or a polynucleotide of anotherorganism, such as a coding or a non-coding sequence, and it may includesingle-stranded or double-stranded DNA molecules, such as a cDNA orgenomic DNA, or RNA molecules, such as pre-mRNA, mRNA, tRNA, and rRNA.The target nucleic acid of interest may be associated with a protospaceradjacent motif (PAM) sequence, which may include a 2-5 base pairsequence adjacent to the protospacer. In some embodiments 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more target nucleic acids can be detected by thedisclosed method.

As used herein, the terms “trans-cleavage”, “trans-endonucleaseactivity”, “trans-mediated endonuclease activity”, “trans-nucleaseactivity”, “trans-mediated nuclease activity” and variations thereofrefer to indiscriminate, non-sequence-specific cleavage of a nucleicacid molecule by an endonuclease (such as by a Cas12, Cas13, and Cas14)which is triggered by cis-(sequence-specific) cleavage. Trans-cleavageis a “multiple turn-over” event, in that more than one substratemolecule is cleaved after initiation by a single turn over cis-cleavageevent.

Type V CRISPR/Cas nucleic acid-guided nucleases are a subtype of Class 2CRISPR/Cas effector nucleases such as, but not limited to, engineeredCas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e),CasY (Cas12d), Cas13a nucleases or naturally-occurring proteins, such asa Cas12a isolated from, for example, Francisella tularensis subsp.novicida (Gene ID: 60806594), Candidatus Methanoplasma termitum (GeneID: 24818655), Candidatus Methanomethylophilus alvus (Gene ID:15139718), and Eubacterium eligens ATCC 27750 (Gene ID: 41356122), andan artificial polypeptide, such as a chimeric protein.

The term “variant” refers to a polypeptide or polynucleotide thatdiffers from a reference polypeptide or polynucleotide but retainsessential properties. A typical variant of a polypeptide differs inamino acid sequence from another reference polypeptide. Generally,differences are limited so that the sequences of the referencepolypeptide and the variant are closely similar overall and, in many ifnot most regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more modifications (e.g.,substitutions, additions, and/or deletions). A variant of a polypeptidemay be a conservatively modified variant. A substituted or insertedamino acid residue may or may not be one encoded by the genetic code(e.g., a non-natural amino acid). A variant of a polypeptide may benaturally occurring, such as an allelic variant, or it may be a variantthat is not known to occur naturally. Variants includemodifications-including chemical modifications—to one or more aminoacids that do not involve amino acid substitutions, additions ordeletions.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, synthetic chromosomes, and the like.

DETAILED DESCRIPTION

The present disclosure provides compositions of matter and cascade assaymethods for detecting nucleic acids where the compositions of matterallow for the reaction kinetics of the cascade assay to be adjusted or“tuned.” The compositions and methods provide for massive multiplexing,high accuracy, low cost, minimum workflow, with results in someembodiments virtually instantaneously, even at ambient temperatures of16-25° C. or less, or, if desired, with slower reaction times but withthe ability to quantify the target nucleic acids of interest withexquisite accuracy.

The cascade assays described herein comprise first and secondribonucleoprotein complexes and either blocked nucleic acid molecules orblocked primer molecules. The blocked nucleic acid molecules or blockedprimer molecules keep the second ribonucleoprotein complexes “locked”unless and until a target nucleic acid of interest activates the firstribonucleoprotein complex, and the molecular design or configuration ofthe blocked nucleic acid molecules (or blocked primer molecules) confersthe “tunability” to the cascade assay. Again, by “locked” it is meantthat the blocked nucleic acid molecules or blocked primer molecules aredesigned in such a way that they are largely blocked from interactingwith the ribonucleoprotein complexes; therefore, the ribonucleoproteincomplexes remain largely inactive (i.e., “locked”) unless and until atarget nucleic acid of interest activates the first ribonucleoproteincomplex The methods comprise the steps of providing cascade assaycomponents, contacting the cascade assay components with a sample, anddetecting a signal that is generated only when a target nucleic acid ofinterest is present in the sample.

Early and accurate identification of, e.g., infectious agents, microbecontamination, variant nucleic acid sequences that indicate the presenceof such diseases such as cancer or contamination by heterologous sourcesis important in order to select correct treatment; identify taintedfood, pharmaceuticals, cosmetics and other commercial goods; and tomonitor the environment. However, currently available state-of-the-artnucleic acid detection such as quantitative PCR (also known as real timePCR or qPCR) relies on DNA amplification, which requires time and maylead to changes to the relative proportion of nucleic acids,particularly in multiplexed nucleic acid assays. The lack of rapidityfor qPCR assays is due to the fact that there is a significant lag phaseearly in the amplification process where fluorescence above backgroundcannot be detected. That is, there is a lag until the cycle threshold orCt value, which is the number of amplification cycles required for thefluorescent signal to exceed the background level of fluorescence, isachieved and can be quantified.

The present disclosure describes a signal boost cascade assay andimprovements thereto that can detect one or more target nucleic acids ofinterest (e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower)limits without the need for amplifying the target nucleic acid(s) ofinterest, thereby avoiding the drawbacks of multiplex amplification,such as primer-dimerization. In addition, the cascade assay is tunable,such that in some embodiments detection of target nucleic acids ofinterest can happen virtually instantaneously, or, alternatively, over alonger period of time. Additionally, the cascade assay can be tuned—viavarying the molecular configuration of the blocked nucleic acidmolecules or blocked primer molecules—to quantify the target nucleicacids of interest over a desired range of concentration; thus providingflexibility for virtually any application. As described in detail below,the cascade assays utilize signal amplification mechanisms comprisingvarious components including nucleic acid-guided nucleases, guide RNAs(gRNAs) incorporated into ribonucleoprotein complexes (RNP complexes),blocked nucleic acid molecules or blocked primer molecules, reportermoieties, and, in some embodiments, polymerases and template moleculeswhere the polymerases copy but do not amplify the template molecules. Aparticularly advantageous feature of the cascade assay is that, with theexception of the gRNA (gRNA1) in RNP1, the cascade assay components canbe essentially identical no matter what target nucleic acid(s) ofinterest are being detected, and gRNA1 is easily programmable. Further,in the context of tunability, the cascade assay is tunable by use ofdifferent blocked nucleic acid molecules (or blocked primer molecules)used to activate RNP2 (described in detail below).

The improvement to the signal amplification or signal boost cascadeassay described herein is drawn to being able to “tune” the cascadeassay by employing differently configured blocked nucleic acid molecules(or blocked primer molecules) that activate RNP2. The present disclosuredemonstrates that by altering the Gibbs free energy (i.e., molecularconfiguration and composition) of the blocked nucleic acid molecules (orblocked primer molecules) employed in the cascade assay, the kinetics ofthe cascade assay can be “tuned.”

FIG. 1A provides a simplified diagram demonstrating a prior art methodfor quantifying target nucleic acids of interest in a sample; namely,the quantitative polymerase chain reaction or qPCR, which to date may beconsidered the gold standard for quantitative detection assays. Thedifference between PCR and qPCR is that PCR is a qualitative techniquethat indicates the presence or absence of a target nucleic acid ofinterest in a sample, where qPCR allows for quantification of targetnucleic acids of interest in a sample. qPCR involves selectiveamplification and quantitative detection of specific regions of DNA orcDNA (i.e., the target nucleic acid of interest) using oligonucleotideprimers that flank the specific region(s) in the target nucleic acid(s)of interest. The primers are used to amplify the specific regions usinga polymerase. Like PCR, repeated cycling of the amplification processleads to an exponential increase in the number of copies of theregion(s) of interest; however, unlike traditional PCR, the increase istracked using an intercalating dye or, as shown in FIG. 1A, asequence-specific probe (e.g., a “Taq-man probe”) the fluorescence ofwhich is detected in real time. RT-qPCR differs from qPCR in that areverse transcriptase is used to first copy RNA molecules to producecDNA before the qPCR process commences.

FIG. 1A is an overview of a qPCR assay where target nucleic acids ofinterest from a sample are amplified before detection. FIG. 1A shows theqPCR method (10), comprising a double-stranded DNA template (12) and asequence-specific Taq-man probe (14) comprising a region complementaryto the target nucleic acid of interest (20), a quencher (16), a quenchedfluorophore (18) where (22) denotes quenching between the quencher (18)and quenched fluorophore (16). Upon denaturation, the two strands of thedouble-stranded DNA template (12) separate into complementary singlestrands (26) and (28). In the next step, primers (24) and (24′), annealto complementary single strands (26) and (28), as does thesequence-specific Taq-man probe (14) via the region complementary (20)to complementary strand (26). Initially the Taq-man probe is annealed tocomplementary strand (26) of the target region of interest intact;however, primers (24) and (24′) are extended by polymerase (30) forminga complement (32) of complementary strand (26); however, the Taq-manprobe is not, due to the absence of a 3′ hydroxy group. Instead, theexonuclease activity of the polymerase “chews up” the Taq-man probe,thereby separating the quencher (16) from the quenched fluorophore (18)resulting in an unquenched or excited-state fluorophore (34). Thefluorescence quenching ensures that fluorescence occurs only when targetnucleic acids of interest are present and being copied, where thefluorescent signal is proportional to the number of single strand targetnucleic acids being amplified.

As noted above, one downside to currently available detection assays isthat they rely on DNA amplification, which, in addition to issues withmultiplexing, significantly hinders the ability to perform rapidtesting, e.g., in the field, where the present cascade assay works atambient temperatures, including room temperature and below. Assays thatrequire amplification of the target nucleic acids of interest do notwork well at lower temperatures—even those assays utilizing isothermalamplification—due to non-specific binding of the primers and lowpolymerase activity. Further, primer design is far more challenging. Asfor the lack of rapidity of qPCR, a significant lag phase occurs earlyin the amplification process where fluorescence above background cannotbe detected, particularly in samples with very low copy numbers of thetarget nucleic acid of interest. And, again, amplification, particularlymultiplex amplification, may cause changes to the relative proportion ofnucleic acids in samples that, in turn, lead to artifacts or inaccurateresults.

A second downside to PCR is that reaction kinetics are defined by primerbinding efficiency and the rate of primer extension by the polymerase.The reaction temperature for a PCR reaction is typically equal to theT_(m) of the primer plus 5° C. This temperature cannot be alteredsignificantly without either decreasing the amount of primer that bindsthe target nucleic acids of interest or increasing non-specific ormis-priming events. Thus, essentially qPCR cannot be tuned to varyreaction time or to quantify target nucleic acids of interest within aspecific window. Another downside to PCR includes complex temperaturecycling (e.g., 95° C. for denaturing, at least 5° C. below T_(m) forannealing, and at least 5° C. above T_(m) for extension), which in turnis dependent on the PCR reagents (e.g., primer concentration, primerlength, polymerase half-life, and the polymerase's rate ofpolymerization).

FIG. 1B provides a simplified diagram demonstrating a prior art method(51) of a CRISPR-based nucleic acid-guided nuclease detection assaywhere target nucleic acids of interest from a sample must be amplifiedin order to be detected, which, like qPCR is not tunable kineticallyexcept via reaction temperature. First, assuming the presence of atarget nucleic acid of interest in a sample, the target nucleic acid ofinterest (52) is amplified to produce many copies of the target nucleicacid of interest (54). The detection assay is initiated (step 2) whenthe target nucleic acid of interest (54) is combined with and binds to apre-assembled ribonucleoprotein complex (56), which is part of areaction mixture. The ribonucleoprotein complex (56) comprises a guideRNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA isintegrated with the nucleic acid-guided nuclease. The gRNA, whichincludes a sequence complementary to the target nucleic acid ofinterest, guides the RNP complex to the target nucleic acid of interestand hybridizes to it, thereby activating the ribonucleoprotein complex(58). The nucleic acid-guided nuclease exhibits (i.e., possesses) bothcis- and trans-cleavage activity, where trans-cleavage activity isinitiated after cis-cleavage activity, or at least upon specific bindingof N nucleotide bases of a target nucleic acid molecule to theribonucleoprotein complex. Cis-cleavage activity occurs as the targetnucleic acid of interest binds to the gRNA and is cleaved by the nucleicacid-guided nuclease (i.e., activation). Once an initial cis-cleavage ofthe target nucleic acid of interest is completed, trans-cleavageactivity is triggered, where trans-cleavage activity is anindiscriminate, non-sequence-specific, and multi-turnover cleavage eventof nucleic acid molecules in the sample.

In step 3, the trans-cleavage activity triggers activation of reportermoieties (62) that are present in the reaction mixture. The reportermoieties (62) may be a synthetic molecule linked or conjugated to aquencher (64) and a fluorophore (66) such as, for example, a probe witha dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′end. The quencher (64) and fluorophore (66) typically are about 20-30bases apart or less for effective quenching via fluorescence resonanceenergy transfer (FRET). Reporter moieties (62) are described in greaterdetail below. As more ribonucleoprotein complexes (56) are activated(56→58), more trans-cleavage activity of the nucleic acid-guidednuclease in the ribonucleoprotein complex is activated and more reportermoieties (68) are activated (where here, “activated” means unquenched);thus, the binding of the target nucleic acid of interest (54). Thesignal change (70) increases as more reporter moieties (68) areactivated.

As noted above, the downside to currently available nucleic acid-guidednuclease detection assays is that they rely on DNA amplification, which,in addition to issues with multiplexing, significantly hinders theability to perform rapid point-of-care testing. The lack of rapidity is,at least in-part, due to cis-cleavage of a target nucleic acid ofinterest being a single turnover event in which the number of activatedenzyme complexes is, at most, equal to the number of copies of thetarget nucleic acids of interest in the sample; thus, PCR amplificationaffects the rapidity of currently available nucleic acid-guided nucleasedetection systems. Once the ribonucleoprotein complex is activated aftercompletion of cis-cleavage, trans-cleavage activity of the reportermoieties that are initially quenched is generated. However, the turnover(K) of, e.g., activated Cas12a complex is 17/sec and 3/sec for dsDNA andssDNA targets, respectively. Therefore, for less than 10,000 targetcopies, the number of reporters cleaved is not sufficient to generate asignal in less than 30-60 minutes.

Thus, like qPCR, a typical CRISPR-based nucleic acid-guided nucleasedetection assay cannot be tuned to vary reaction times or to quantifytarget nucleic acids of interest over a specific concentration window.In contrast, the cascade assay described herein which utilizes tworibonucleoprotein (RNP) complexes can be tuned, allowing for maximumflexibility. FIG. 1C provides a simplified diagram demonstrating amethod (100) of a cascade assay. The cascade assay is initiated when thetarget nucleic acid of interest (104) binds to and activates a firstpre-assembled ribonucleoprotein complex (RNP1) (102). Aribonucleoprotein complex comprises a guide RNA (gRNA) and a nucleicacid-guided nuclease, where the gRNA is integrated with the nucleicacid-guided nuclease. The gRNA, which includes a sequence complementaryto the target nucleic acid of interest, guides an RNP complex to thetarget nucleic acid of interest and hybridizes to it. Typically,preassembled RNP complexes are employed in the reaction mixture—asopposed to separate nucleic acid-guided nucleases and gRNAs—tofacilitate rapid (virtually instantaneous) detection of the targetnucleic acid(s) of interest, if desired.

“Activation” of RNP1 (106) in the context of the cascade assay refers toactivating trans-cleavage activity of the nucleic acid-guided nucleasein RNP1 (106) by first initiating cis-cleavage where the target nucleicacid of interest is cleaved by the nucleic acid-guided nuclease, or atleast upon specific binding of N nucleotide bases of a target nucleicacid molecule to the ribonucleoprotein complex. This cis-cleavageactivity then initiates trans-cleavage activity (i.e., multi-turnoveractivity) of the nucleic acid-guided nuclease, where trans-cleavage isindiscriminate, leading to non-sequence-specific cutting of nucleic acidmolecules by the nucleic acid-guided nuclease of RNP1 (102). Thistrans-cleavage activity triggers activation of blocked ribonucleoproteincomplexes (RNP2s) (108) via blocked nucleic acid molecules (or in analternative embodiment, blocked primer molecules), which are describedin detail below. Each newly activated RNP2 (110) activates more RNP2(108→110), which in turn cleave reporter moieties (112). The reportermoieties (112) may be a synthetic molecule linked or conjugated to aquencher (114) and a fluorophore (116) such as, for example, a probewith a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the3′ end. The quencher (114) and fluorophore (116) can be about 20-30bases apart or less for effective quenching via fluorescence resonanceenergy transfer (FRET). Reporter moieties may also be incorporated intoblocked nucleic acid molecules or blocked primer molecules—which alsoaffects the kinetics of the cascade assay reaction—and are described ingreater detail below.

As more RNP2s are activated (108→110), more trans-cleavage activity isactivated and more reporter moieties (118) are unquenched; thus, thebinding of the target nucleic acid of interest (104) to RNP1 (102)initiates what becomes a cascade of signal production (120), whichincreases exponentially, hence, the terms signal amplification or signalboost. The cascade assay thus comprises a single turnover event thattriggers a multi-turnover event that then triggers anothermulti-turnover event. As described below in relation to FIG. 4 , thereporter moieties (112) may be provided as molecules that are separatefrom the other components of the nucleic acid-guided nuclease cascadeassay, or the reporter moieties may be covalently or non-covalentlylinked to the blocked nucleic acid molecules or synthesized activatingmolecules (i.e., the target molecules for the RNP2). As described indetail below, the present description presents blocked nucleic acidmolecules, which can be “tuned” to provide varying reaction kinetics forthe cascade assay.

Target Nucleic Acids of Interest

The target nucleic acid of interest may be a DNA, RNA, or cDNA molecule.Target nucleic acids of interest may be isolated from a sample ororganism by standard laboratory techniques or may be synthesized bystandard laboratory techniques (e.g., RT-PCR). The target nucleic acidsof interest are identified in a sample, such as a biological sample froma subject (including non-human animals or plants), items of manufacture,or an environmental sample (e.g., water or soil). Non-limiting examplesof biological samples include blood, serum, plasma, saliva, mucus, anasal swab, a buccal swab, a cell, a cell culture, and tissue. Thesource of the sample could be any mammal, such as, but not limited to, ahuman, primate, monkey, cat, dog, mouse, pig, cow, horse, sheep (andother livestock), and bat. Samples may also be obtained from any othersource, such as air, water, soil, surfaces, food, beverages,nutraceuticals, clinical sites or products, industrial sites andproducts, plants and grains, cosmetics, personal care products,pharmaceuticals, medical devices, agricultural equipment and sites, andcommercial samples.

In some embodiments, the target nucleic acid of interest is from aninfectious agent (e.g., a bacteria, protozoan, insect, worm, virus, orfungus) that affects mammals. As a non-limiting example, the targetnucleic acid of interest could be one or more nucleic acid moleculesfrom bacteria, such as Bordetella parapertussis, Bordetella pertussis,Chlamydia pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae,Acinetobacter calcoaceticus-baumannii complex, Bacteroides fragilis,Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes,Klebsiella oxytoca, Klebsiella pneumoniae group, Moraxella catarrhalis,Proteus spp., Salmonella enterica, Serratia marcescens, Haemophilusinfluenzae, Neisseria meningitides, Pseudomonas aeruginosa,Stenotrophomonas maltophilia, Enterococcus faecalis, Enterococcusfaecium, Listeria monocytogenes, Staphylococcus aureus, Staphylococcusepidermidis, Staphylococcus lugdunensis, Streptococcus agalactiae,Streptococcus pneumoniae, Streptococcus pyogenes, Chlamydia tracomatis,Neisseria gonorrhoeae, Syphilis (Treponema pallidum), Ureaplasmaurealyticum, Mycoplasma genitalium, and/or Gardnerella vaginalis.

As a non-limiting example, the target nucleic acid of interest could beone or more nucleic acid molecules from a virus, such as adenovirus,coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus 0C43,severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), humanmetapneumovirus, human rhinovirus, enterovirus, influenza A, influenzaA/H1, influenza A/H3, influenza A/H1-2009, influenza B, parainfluenzavirus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenzavirus 4, respiratory syncytial virus, herpes simplex virus 1, herpessimplex virus 2, human immunodeficiency virus (HIV), humanpapillomavirus, hepatitis A virus (HAV), hepatitis B virus (HBV),hepatitis C virus (HCV), and/or human parvovirus B19 (B19V).

Also, as a non-limiting example, the target nucleic acid of interestcould be one or more nucleic acid molecules from a fungus, such asCandida albicans, Candida auris, Candida glabrata, Candida krusei,Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans,and/or Cryptococcus gattii. As another non-limiting example, the targetnucleic acid of interest could be one or more nucleic acid moleculesfrom a protozoan, such as Trichomonas vaginalis, Bonamia exitiosa,Bonamia ostreae, Leishmania amazonensis, Leishmania braziliensis,Leishmania donovani, Leishmania infantum, Leishmania major, Leishmaniamexicana, Leishmania tropica, Marteilia refringens, Perkinsus marinus,Perkinsus olseni, Theileria annulata, Theileria equi, Theileria parva,Tritrichomonas foetus, Trypanosoma brucei, Trypanosoma congolense,Trypanosoma equiperdum, Trypanosoma evansi and, Trypanosoma vivax.

Additionally, the target nucleic acid of interest may originate in anorganism such as a bacterium, virus, fungus or other pest that infectslivestock or agricultural crops. Such organisms include avian influenzaviruses, mycoplasma and other bovine mastitis pathogens, Clostridiumperfringens, Campylobacter sp., Salmonella sp., Pospirivoidae,Avsunvirodiae, Panteoea stewartii, Mycoplasma genitalium, Sprioplasmasp., Pseudomonas solanacearum, Erwinia amylovora, Erwinia carotovora,Pseudomonas syringae, Xanthomonas campestris, Agrobacterium tumefaciens,Spiroplasma citri, Phytophthora infestans, Endothia parasitica,Ceratocysis ulmi, Puccinia graminis, Hemilea vastatrix, Ustilage maydis,Ustilage nuda, Guignardia bidwellii, Uncinula necator, Botrytiscincerea, Plasmopara viticola, or Botryotinis fuckleina.

In some embodiments, other target nucleic acids of interest may be fornon infectious conditions, e.g., to be used for genotyping, includingnon-invasive prenatal diagnosis of, e.g, trisomies, other chromosomalabnormalities, and known genetic diseases such as Tay Sachs disease andsickle cell anemia. Other target nucleic acids of interest and samplesare described herein. Target nucleic acids of interest may includeengineered biologics, including cells such as chimeric antigen receptorT (CAR-T) cells, or target nucleic acids of interest from very small orrare samples, where only small volumes are available for testing.

The cascade assays described herein are particularly well-suited forsimultaneous testing of multiple targets. Pools of two to 10,000 targetnucleic acids of interest may be employed, e.g., 2-1000, 2-100, 2-50, or2-10. Further testing may be used to identify the specific member of thepool, if warranted.

While the methods described herein do not require the target nucleicacid of interest to be DNA (and in fact it is specifically contemplatedthat the target nucleic acid of interest may be RNA), it is understoodby those in the field that a reverse transcription step to converttarget RNA to cDNA may be performed prior to or while contacting thebiological sample with the composition. Alternatively, RNA targetnucleic acids of interest can be detected directly via RNA-specificnucleic acid nucleases such as Cas13a or Cas12g.

Nucleic Acid-Guided Nucleases

The cascade assays comprise nucleic acid-guided nucleases in thereaction mixture, either provided as a protein, a coding sequence forthe protein, or, in many embodiments, in a pre-assembledribonucleoprotein (RNP) complex. In some embodiments, the one or morenucleic acid-guided nucleases in the reaction mixture may be, forexample, a Cas endonuclease. Any nucleic acid-guided nuclease havingboth cis- and trans-endonuclease activity may be employed, and the samenucleic acid-guided nuclease may be used for both RNP complexes ordifferent nucleic acid-guided nucleases may be used in RNP1 and RNP2.Note that trans-cleavage activity is not triggered unless and untilcis-cleavage activity (i.e., sequence-specific activity) is initiated.Nucleic acid-guided nucleases include Type V and Type VI nucleicacid-guided nucleases, as well as nucleic acid-guided nucleases thatcomprise a RuvC nuclease domain or a RuvC-like nuclease domain but lackan HNH nuclease domain. Nucleic acid-guided nucleases with theseproperties are reviewed in Makarova and Koonin, Methods Mol. Biol.,1311:47-75 (2015) and Koonin, et al., Current Opinion in Microbiology,37:67-78 (2020) and updated databases of nucleic acid-guided nucleasesand nuclease systems that include newly-discovered systems includeBioGRID ORCS (orcs:thebiogrid.org); GenomeCRISPR (genomecrispr.org);Plant Genome Editing Database (plantcrispr.org) and CRISPRCasFinder(crispercas.i2bc.paris-saclay.fr).

The type of nucleic acid-guided nuclease utilized in the method ofdetection depends on the type of target nucleic acid of interest to bedetected. For example, a DNA nucleic acid-guided nuclease (e.g., aCas12a, Cas14a, or Cas3) should be utilized if the target nucleic acidof interest is a DNA molecule, and an RNA nucleic acid-guided nuclease(e.g., Cas13a or Cas12g) should be utilized if the target nucleic acidof interest is an RNA molecule. Exemplary nucleic acid-guided nucleasesinclude, but are not limited to, Cas RNA-guided DNA endonucleases, suchas Cas3, Cas12a (e.g., AsCas12a, LbCas12a), Cas12b, Cas12c, Cas12d,Cas12e, Cas14, Cas12h, Cas12i, and Cas12j; Cas RNA-guided RNAendonucleases, such as Cas13a (LbaCas13, LbuCas13, LwaCas13), Cas13b(e.g., CccaCas13b, PsmCas13b), and Cas12g; and any other nucleic acid(DNA, RNA, or cDNA) targeting nucleic acid-guided nuclease withcis-cleavage activity and collateral trans-cleavage activity. In someembodiments, the nucleic acid-guided nuclease is a Type V CRISPR-Casnuclease, such as a Cas12a, Cas13a, or Cas14a. In some embodiments, thenucleic acid-guided nuclease is a Type I CRISPR-Cas nuclease, such asCas3. Type II and Type VI nucleic acid-guided nucleases may also beemployed.

Guide RNA (gRNA)

The present disclosure detects a target nucleic acid of interest via areaction mixture containing at least two guide RNAs (gRNAs) eachincorporated into an RNP complex (i.e., RNP1 or RNP2). Suitable gRNAsinclude at least one crRNA region to enable specificity in everyreaction. The gRNA of RNP1 is specific to a target nucleic acid ofinterest and the gRNA of RNP2 is specific to an unblocked nucleic acidor a synthesized activating molecule (both described in detail below).As will be clear given the description below, an advantageous feature ofthe cascade assay is that, with the exception of the gRNA in the RNP1(i.e., the gRNA specific to the target nucleic acid of interest), thecascade assay components can stay the same (i.e., are identical orsubstantially identical) no matter what target nucleic acid(s) ofinterest are being detected, and the gRNA in RNP1 is easilyreprogrammable. In the context of tunability, the cascade assay istunable by use of blocked nucleic acid molecules or blocked primermolecules having various molecular configurations (i.e., free energies).Once desired reaction kinetics and/or a target quantification window isidentified, this particular version of the cascade assay can bereprogrammed by changing the gRNA in RNP1.

Like the nucleic acid-guided nuclease, the gRNA may be provided in thecascade assay reaction mixture in a preassembled RNP, as an RNAmolecule, or may also be provided as a DNA sequence to be transcribed,in, e.g., a vector backbone. Providing the gRNA in a pre-assembled RNPcomplex (i.e., RNP1 or RNP2) is preferred if rapid assay kinetics arepreferred. If provided as a gRNA molecule, the gRNA sequence may includemultiple endoribonuclease recognition sites (e.g., Csy4) for multiplexprocessing. Alternatively, if provided as a DNA sequence to betranscribed, an endoribonuclease recognition site is encoded betweenneighboring gRNA sequences and more than one gRNA can be transcribed ina single expression cassette. Direct repeats can also serve asendoribonuclease recognition sites for multiplex processing. Guide RNAsare generally about 20 nucleotides to about 300 nucleotides in lengthand may contain a spacer sequence containing a plurality of bases andcomplementarity to a protospacer sequence in the target sequence. ThegRNA spacer sequence may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%,98%, 99%, or more complementary to its intended target nucleic acid ofinterest.

The gRNA of RNP1 is capable of complexing with the nucleic acid-guidednuclease of RNP1 to perform cis-cleavage of a target nucleic acid ofinterest (i.e., a DNA or RNA), which triggers non-sequence-specifictrans-cleavage of other molecules in the reaction mixture. Guide RNAsinclude any polynucleotide sequence having sufficient complementaritywith a target nucleic acid of interest (or target sequences generated byunblocking blocked nucleic acid molecules or target sequences generatedby synthesizing activating molecules as described below). Target nucleicacids of interest may include a protospacer-adjacent motif (PAM), and,following gRNA binding, the nucleic acid-guided nuclease induces adouble-stranded break either inside or outside the protospacer region ofthe target nucleic acid of interest.

In some embodiments, the gRNA (e.g., of RNP1) is an exo-resistantcircular molecule that can include several DNA bases between the 5′ endand the 3′ end of a natural guide RNA and is capable of binding a targetsequence. The length of the circularized guide for RNP1 can be such thatthe circular form of guide can be complexed with a nucleic acid-guidednuclease to form a modified RNP1 which can still retain its cis-cleavagei.e., (specific) and trans-cleavage (i.e., non-specific) nucleaseactivity.

In any of the foregoing embodiments, the gRNA may be a modified ornon-naturally occurring nucleic acid molecule. In some embodiments, thegRNAs of the disclosure may further contain a locked nucleic acid (LNA),a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). Byway of further example, a modified nucleic acid molecule may contain amodified or non-naturally occurring nucleoside, nucleotide, and/orinternucleoside linkage, such as a 2′-O-methyl (2′-O-Me) modifiednucleoside, a 2′-fluoro (2′-F) modified nucleoside, and aphosphorothioate (PS) bond, or any other nucleic acid moleculemodifications described herein.

Ribonucleoprotein (RNP) Complex

As described above, although the assay “reaction mixture” may compriseseparate nucleic acid-guided nucleases and gRNAs (or coding sequencestherefor), the cascade assays preferably comprise preassembledribonucleoprotein complexes (RNPs) in the reaction mixture, allowing forfaster detection kinetics. The present cascade assay employs at leasttwo types of RNP complexes, RNP1 and RNP2, each type containing anucleic acid-guided nuclease and a gRNA. RNP1 and RNP2 may comprise thesame nucleic acid-guided nuclease or may comprise different nucleicacid-guided nucleases; however, the gRNAs in RNP1 and RNP2 are differentand are configured to detect different nucleic acids. In someembodiments, the reaction mixture contains about 1 fM to about 10 μM ofa given RNP1, or about 1 pM to about 1 μM of a given RNP1, or about 10pM to about 500 pM of a given RNP1. In some embodiments the reactionmixture contains about 6×10⁴ to about 6×10¹² complexes per microliter(μl) of a given RNP1, or about 6×10⁶ to about 6×10¹⁰ complexes permicroliter (μl) of a given RNP1. In some embodiments, the reactionmixture contains about 1 fM to about 500 μM of a given RNP2, or about 1pM to about 250 μM of a given RNP2, or about 10 pM to about 100 μM of agiven RNP2. In some embodiments the reaction mixture contains about6×10⁴ to about 6×10¹² complexes per microliter (μl) of a given RNP2 orabout 6×10⁶ to about 6×10¹² complexes per microliter (μl) of a givenRNP2.

In any of the embodiments of the disclosure, the reaction mixtureincludes 1 to about 1,000 different RNP1s (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 19, 20, 21, 22, 23, 24, 25, 50,75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, or 1,0000 RNP1s), where differentRNP1s comprise a different gRNA (or crRNA thereof) polynucleotidesequence. For example, a reaction mixture designed for environmental oroncology testing comprises more than one unique RNP1-gRNA (orRNP1-crRNA) ribonucleoprotein complex for the purpose of detecting morethan one target nucleic acid of interest. That is, more than one RNP1may be present for the purpose of targeting one target nucleic acid ofinterest from many sources or more than one RNP1 may be present fortargeting more than one target nucleic acid of interest from a singleorganism or condition.

In any of the foregoing embodiments, the gRNA of RNP1 may be homologousor heterologous, relative to the gRNA of other RNP1(s) present in thereaction mixture. A homologous mixture of RNP1 gRNAs has a number ofgRNAs with the same nucleotide sequence, whereas a heterologous mixtureof RNP1 gRNAs has multiple gRNAs with different nucleotide sequences(e.g., gRNAs targeting different loci, genes, variants, and/or microbialspecies). Therefore, the disclosed methods of identifying one or moretarget nucleic acids of interest may include a reaction mixturecontaining more than two heterologous gRNAs, more than threeheterologous gRNAs, more than four heterologous gRNAs, more than fiveheterologous gRNAs, more than six heterologous gRNAs, more than sevenheterologous gRNAs, more than eight heterologous gRNAs, more than nineheterologous gRNAs, more than ten heterologous gRNAs, more than elevenheterologous gRNAs, more than twelve heterologous gRNAs, more thanthirteen heterologous gRNAs, more than fourteen heterologous gRNAs, morethan fifteen heterologous gRNAs, more than sixteen heterologous gRNAs,more than seventeen heterologous gRNAs, more than eighteen heterologousgRNAs, more than nineteen heterologous gRNAs, more than twentyheterologous gRNAs, more than twenty-one heterologous gRNAs, more thantwenty-three heterologous gRNAs, more than twenty-four heterologousgRNAs, or more than twenty-five heterologous gRNAs. Such a heterologousmixture of RNP1 gRNAs in a single reaction enables multiplex testing.

As a first non-limiting example of a heterologous mixture of RNP1 gRNAs,the reaction mixture may contain: a number of RNP1s having a gRNAtargeting parainfluenza virus 1; a number of RNP1 s having a gRNAtargeting human metapneumovirus; a number of RNP1s having a gRNAtargeting human rhinovirus; a number of RNP1 s having a gRNA targetinghuman enterovirus; a number of RNP1 having a gRNA targeting respiratorysyncytial virus; and a number of RNP1s having a gRNA targetingcoronavirus HKU1. As a second non-limiting example of a heterologousmixture of RNP1 gRNAs, the reaction mixture may contain: a number ofRNP1 s containing a gRNA targeting two or more SARS-Co-V-2 variants,e.g., B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1, BA.2, BA.2.12.1, BA.4, andBA.5 and subvariants thereof.

As another non-limiting example of a heterologous mixture of RNP1 gRNAs,the reaction mixture may contain RNP1 s targeting two or more targetnucleic acids of interest from, e.g., organisms that infect vineyards,such as Guignardia bidwellii, Uncinula necator, Botrytis cincerea,Plasmopara viticola, and Botryotinis fuckleina.

Reporter Moieties

The cascade assay detects a target nucleic acid of interest viadetection of a signal generated in the reaction mixture by a reportermoiety. In some embodiments the detection of the target nucleic acid ofinterest occurs virtually instantaneously at 3M or 30 copies and within1 minute or less at 3 copies (see, e.g., FIGS. 6B-6H).

Depending on the type of reporter moiety used, trans- and/orcis-cleavage by the nucleic acid-guided nuclease in RNP2 releases asignal. In some embodiments, trans-cleavage of stand-alone (e.g., notbound to any blocked nucleic acid molecules) reporter moieties maygenerate signal changes at rates that are proportional to the cleavagerate, as new RNP2s are activated over time (shown at bottom in FIGS. 2A,3A and 3B). Trans-cleavage by either an activated RNP1 or an activatedRNP2 may release a signal; thus, when the reporter moiety is a separatemolecule, the reporter moieties are activated quickly by thetrans-cleavage activity. The reporter moiety can comprise DNA, RNA, achimera of DNA and RNA, or an oligonucleotide with modified nucleicacids. The reporter moiety also can comprise both single- anddouble-stranded portions.

In alternative embodiments and preferably, the reporter moiety may bebound to the blocked nucleic acid molecule, where trans-cleavage of theblocked nucleic acid molecule and conversion to an unblocked nucleicacid molecule may generate signal changes at rates that are proportionalto the cleavage rate, as new RNP2s are activated over time, thusallowing for real time reporting of results (shown at FIG. 4 , center).In this embodiment, the reaction kinetics of signal generation matchthat of the cascade assay reaction rate. The signal is generated as theblocked nucleic acid molecule is unblocked, whether quickly or slowly.In yet another embodiment, the reporter moiety may be bound to a blockednucleic acid molecule such that cis-cleavage following the binding ofthe RNP2 to an unblocked nucleic acid molecule releases a PAM distalsequence, which in turn generates a signal at rates that areproportional to the cleavage rate (shown at FIG. 4 , bottom). In thiscase, activation of RNP2 by cis-(target specific) cleavage of theunblocked nucleic acid molecule directly produces a signal, rather thanproducing a signal via indiscriminate trans-cleavage activity.Alternatively or in addition, the reporter moiety may be bound to thegRNA.

The reporter moiety may be a synthetic molecule linked or conjugated toa reporter and quencher such as, for example, a TAQMAN® probe with a dyelabel (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end.The reporter and quencher may be about 20-30 bases apart or less foreffective quenching via fluorescence resonance energy transfer (FRET).Alternatively, signal generation may occur through different mechanisms.Other detectable moieties, labels, or reporters can also be used todetect a target nucleic acid of interest as described herein. Reportermoieties can be labeled in a variety of ways, including direct orindirect attachment of a detectable moiety such as a fluorescent moiety,hapten, or colorimetric moiety.

Examples of detectable moieties include various radioactive moieties,enzymes, prosthetic groups, fluorescent markers, luminescent markers,bioluminescent markers, metal particles, and protein-protein bindingpairs, e.g., protein-antibody binding pairs. Examples of fluorescentmoieties include, but are not limited to, yellow fluorescent protein(YFP), green fluorescence protein (GFP), cyan fluorescence protein(CFP), umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, cyanines, dansylchloride, phycocyanin, and phycoerythrin. Examples of bioluminescentmarkers include, but are not limited to, luciferase (e.g., bacterial,firefly, click beetle and the like), luciferin, and aequorin. Examplesof enzyme systems having visually detectable signals include, but arenot limited to, galactosidases, glucorinidases, phosphatases,peroxidases, and cholinesterases. Identifiable markers also includeradioactive elements such as ¹²⁵I, ³⁵S, ¹⁴C, or ³H. Reporters can alsoinclude a change in pH or charge of the cascade assay reaction mixture.

The methods used to detect the generated signal will depend on thereporter moiety or moieties used. For example, a radioactive label canbe detected using a scintillation counter, photographic film as inautoradiography, or storage phosphor imaging. Fluorescent labels can bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence can bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Enzymatic labels can be detected byproviding the appropriate substrates for the enzyme and detecting theresulting reaction product. Simple colorimetric labels can be detectedby observing the color associated with the label. When pairs offluorophores are used in an assay, fluorophores are chosen that havedistinct emission patterns (wavelengths) so that they can be easilydistinguished. In some embodiments, the signal can be detected bylateral flow assays (LFAs). Lateral flow tests are simple devicesintended to detect the presence or absence of a target nucleic acid ofinterest in a sample. LFAs can use nucleic acid molecules conjugatednanoparticles (often gold, e.g., RNA-AuNPs or DNA-AuNPs) as a detectionprobe, which hybridizes to a complementary target sequence. (See FIG. 5and the description thereof below.) The classic example of an LFA is thehome pregnancy test.

Single-stranded nucleic acid reporter moieties such as ssDNA reportermoieties or RNA molecules can be introduced to show a signal changeproportional to the cleavage rate, which increases with every newactivated RNP2 complex over time. In some embodiments and as describedin detail below, single-stranded nucleic acid reporter moieties can alsobe embedded into the blocked nucleic acid molecules for real timereporting of results.

For example, the method of detecting a target nucleic acid molecule in asample using a cascade assay as described herein can involve contactingthe reaction mixture with a labeled detection ssDNA containing afluorescent resonance energy transfer (FRET) pair, a quencher/phosphorpair, or both. A FRET pair consists of a donor chromophore and anacceptor chromophore, where the acceptor chromophore may be a quenchermolecule. FRET pairs (donor/acceptor) suitable for use include, but arenot limited to, EDANS/fluorescein, IAEDANS/fluorescein,fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL,fluorescein/QSY™ (succinimidyl ester)-7, fluorescein/LC Red 640,fluorescein/Cy 5.5, Texas Red/DABCYL, BODIPY™(4,4-difluoro-4-bora-3A,4A-diaza-s-indacene)/DABCYL, Luciferyellow/DABCYL, coumarin/DABCYL, and fluorescein/LC Red 705. In addition,a fluorophore/quantum dot donor/acceptor pair can be used. EDANS(5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid); IAEDANS is5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid);DABCYL is 4-(4-dimethylaminophenyl) diazenylbenzoic acid. Usefulquenchers include, but are not limited to, DABCYL, QSY™ (succinimidylester) 7 and QSY™ (succinimidyl ester) 33.

In any of the foregoing embodiments, the reporter moiety may compriseone or more modified nucleic acid molecules, containing a modifiednucleoside or nucleotide. In some embodiments the modified nucleoside ornucleotide is chosen from 2′-O-methyl (2′-O-Me) modified nucleoside, a2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond,or any other nucleic acid molecule modifications described below.

Nucleic Acid Modifications

For any of the nucleic acid molecules described herein (e.g., blockednucleic acid molecules, blocked primer molecules, gRNAs, templatemolecules, synthesized activating molecules, and reporter moieties), thenucleic acid molecules may be used in a wholly or partially modifiedform. Typically, modifications to the blocked nucleic acids, gRNAs,template molecules, reporter moieties, and blocked primer moleculesdescribed herein are introduced to optimize the molecule's biophysicalproperties (e.g., increasing endonuclease resistance and/or increasingthermal stability). Modifications typically are achieved by theincorporation of, for example, one or more alternative nucleosides,alternative sugar moieties, and/or alternative internucleoside linkages.

For example, one or more of the cascade assay components may include oneor more of the following nucleoside modifications: 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine and other alkynylderivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, and/or 3-deazaguanine and 3-deazaadenine. The nucleicacid molecules described herein (e.g., blocked nucleic acid molecules,blocked primer molecules, gRNAs, reporter molecules, synthesizedactivating molecules, and template molecules) may also includenucleobases in which the purine or pyrimidine base is replaced withother heterocycles, for example 7-deaza-adenine, 7-deazaguanosine,2-aminopyridine, and/or 2-pyridone. Further modification of the nucleicacid molecules described herein may include nucleobases disclosed inU.S. Pat. No. 3,687,808; Kroschwitz, ed., The Concise Encyclopedia ofPolymer Science and Engineering, New York, John Wiley & Sons, 1990, pp.858-859; Englisch, et al., Angewandte Chemie, 30:613 (1991); andSanghvi, Chapter 16, Antisense Research and Applications, CRC Press,Gait, ed., 1993, pp. 289-302.

In addition to or as an alternative to nucleoside modifications, thecascade assay components may comprise 2′ sugar modifications, including2′-O-methyl (2′-O-Me), 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE), 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, and/or2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylamino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂OCH₂N(CH₃)₂. Other possible 2′-modifications that can modify thenucleic acid molecules described herein (i.e., blocked nucleic acids,gRNAs, synthesized activating molecules, reporter molecules, and blockedprimer molecules) may include all possible orientations of OH; F; O-,S-, or N-alkyl (mono- or di-); O-, S-, or N-alkenyl (mono- or di-); O-,S- or N-alkynyl (mono- or di-); or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkylor C2 to C10 alkenyl and alkynyl. Other potential sugar substituentgroups include, e.g., aminopropoxy (—OCH₂CH₂CH₂NH₂), ally (—CH₂—CH═CH₂),—O-ally (—O—CH₂—CH═CH₂) and fluoro (F). 2′-sugar substituent groups maybe in the arabino (up) position or ribo (down) position. In someembodiments, the 2′-arabino modification is 2′-F. Similar modificationsmay also be made at other positions on the interfering RNA molecule,particularly the 3′ position of the sugar on the 3′ terminal nucleosideor in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminalnucleotide. Oligonucleotides may also have sugar mimetics such ascyclobutyl moieties in place of the pentofuranosyl sugar.

Finally, modifications to the cascade assay components may compriseinternucleoside modifications such as phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates, and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.

The Signal Boosting Cascade Assay Employing Blocked Nucleic Acids

Before getting to the details relating to tuning the kinetics of thecascade assay via the blocked nucleic acid molecules (or blocked primermolecules), understanding the cascade assay itself is key. FIG. 1C,described above, depicts the cascade assay generally. A specificembodiment of the cascade assay utilizing blocked nucleic acids isdepicted in FIG. 2A and described in detail below. In this embodiment, ablocked nucleic acid is used to prevent the activation of RNP2 in theabsence of a target nucleic acid of interest. The method (200) in FIG.2A begins with providing the cascade assay components RNP1 (201), RNP2(202) and blocked nucleic acid molecules (203). RNP1 (201) comprises agRNA specific for a target nucleic acid of interest and a nucleicacid-guided nuclease (e.g., Cas12a or Cas14 for a DNA target nucleicacid of interest or a Cas 13a for an RNA target nucleic acid ofinterest) and RNP2 (202) comprises a gRNA specific for an unblockednucleic acid molecule and a nucleic acid-guided nuclease (again, Cas12aor Cas14 for a DNA unblocked nucleic acid molecule or a Cas13a for anRNA unblocked nucleic acid molecule). As described above, the nucleicacid-guided nucleases in RNP1 (201) and RNP2 (202) can be the same ordifferent depending on the type of target nucleic acid of interest andunblocked nucleic acid molecule. What is key, however, is that thenucleic acid-guided nucleases in RNP1 and RNP2 may be activated to havetrans-cleavage activity following initiation of cis-cleavage activity.

In a first step, a sample comprising a target nucleic acid of interest(204) is added to the cascade assay reaction mixture. The target nucleicacid of interest (204) combines with and activates RNP1 (205) but doesnot interact with or activate RNP2 (202). Once activated, RNP1 cuts thetarget nucleic acid of interest (204) via sequence-specificcis-cleavage, which then activates non-specific trans-cleavage of othernucleic acids present in the reaction mixture, including the blockednucleic acid molecules (203). At least one of the blocked nucleic acidmolecules (203) becomes an unblocked nucleic acid molecule (206) whenthe blocking moiety (207) is removed. As described below, “blockingmoiety” may refer to nucleoside modifications, topographicalconfigurations such as secondary structures, and/or structuralmodifications.

Once at least one of the blocked nucleic acid molecules (203) isunblocked, the unblocked nucleic acid molecule (206) can then interactwith and activate an RNP2 (208). Because the nucleic acid-guidednucleases in the RNP1s (205) and RNP2s (208) have both cis- andtrans-cleavage activity, more blocked nucleic acid molecules (203)become unblocked nucleic acid molecules (206) triggering activation ofmore RNP2s (208) and more trans-cleavage activity in a cascade. FIG. 2Aat bottom depicts the concurrent activation of reporter moieties. Intactreporter moieties (209) comprise a quencher (210) and a fluorophore(211) linked by a nucleic acid sequence. As described above in relationto FIG. 1C, the reporter moieties are also subject to trans-cleavage byactivated RNP1 (205) and RNP2 (208). The intact reporter moieties (209)become activated reporter moieties (212) when the quencher (210) isseparated from the fluorophore (211), emitting a fluorescent signal(213). Signal strength increases rapidly as more blocked nucleic acidmolecules (203) become unblocked nucleic acid molecules (206) triggeringcis-cleavage activation of more RNP2s (208) and thus more trans-cleavageactivity of the reporter moieties (209). Again, here the reportermoieties are shown as separate molecules from the blocked nucleic acidmolecules, but other configurations may be employed and are discussed inrelation to FIG. 4 . One particularly advantageous feature of thecascade assay is that, with the exception of the gRNA in the RNP1(gRNA1), the cascade assay components are modular in the sense that thecomponents stay the same no matter what target nucleic acid(s) ofinterest are being detected. Further, as described below, the cascadeassay is tunable by use of blocked nucleic acid molecules or blockedprimer molecules having different configurations and free energies.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid molecule(220) and an exemplary technique for unblocking the blocked nucleic acidmolecules described herein. A blocked single-stranded ordouble-stranded, circular or linear, DNA or RNA molecule (220)comprising a target strand (222) may contain a partial hybridizationwith a complementary non-target strand nucleic acid molecule (224)containing unhybridized and cleavable secondary loop structures (226)(e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissinghairpins, internal loops, bulges, and multibranch loops). Trans-cleavageof the loops by, e.g., activated RNP1s or RNP2s, generates short strandnucleotide sequences (228) which, because of the short length and lowmelting temperature T_(m), can dehybridize at room temperature (e.g.,15°−25° C.), thereby unblocking the blocked nucleic acid molecule (220)to create an unblocked nucleic acid molecule (230), enabling theinternalization of the unblocked nucleic acid molecule (230) (targetstrand) into an RNP2, leading to RNP2 activation.

A blocked nucleic acid molecule may be single-stranded ordouble-stranded, circular or linear, and may further contain a partiallyhybridized nucleic acid sequence containing cleavable secondary loopstructures, as exemplified by “L” in FIGS. 2C-2E. Such blocked nucleicacids typically have a low binding affinity, or high dissociationconstant (K_(d)) in relation to binding to RNP2 and may be referred toherein as a high K_(d) nucleic acid molecule. In the context of thepresent disclosure, the binding of blocked or unblocked nucleic acidmolecules or blocked primer molecules or synthesized activatingmolecules to RNP2 have low K_(d) values ranging from about 100 fM toabout 1 aM or lower (e.g., 100 zM). High K_(d) values range from 100 nMto about 10-100 10 mM; thus, high K_(d) values are about 10⁵-,10⁶-,10⁷-, 10⁸-, 10⁹-to 10¹⁰-fold or higher as compared to low K_(d)values. Of course, the ideal blocked nucleic acid molecule would have an“infinite K_(d).”

The blocked nucleic acid molecules (high K_(d) molecules) describedherein can be converted into unblocked nucleic acid molecules (low K_(d)molecules—also in relation to binding to RNP2) via cleavage ofnuclease-cleavable regions (e.g., via active RNP1s and RNP2s). Theunblocked nucleic acid molecule has a higher binding affinity for thegRNA in the RNP2 than does the blocked nucleic acid molecule, although,as described below, there may be some “leakiness” where some blockednucleic acid molecules are able to interact with the gRNA in the RNP2.

Once the unblocked nucleic acid molecule is bound to RNP2, the RNP2activation triggers trans-cleavage activity, which in turn leads to moreRNP2 activation by further cleaving blocked nucleic acid molecules toproduce more unblocked nucleic acid molecules, resulting in a positivefeedback loop.

In embodiments where blocked nucleic acid molecules are linear and/orform a secondary structure, the blocked nucleic acid molecules may besingle-stranded (ss) or double-stranded (ds) and contain a firstnucleotide sequence and a second nucleotide sequence. The firstnucleotide sequence has sufficient complementarity to hybridize to agRNA of RNP2, and the second nucleotide sequence does not. The first andsecond nucleotide sequences of a blocked nucleic acid molecule may be onthe same nucleic acid molecule (e.g., for single-strand embodiments) oron separate nucleic acid molecules (e.g., for double strandembodiments). Trans-cleavage (e.g., via RNP1 or RNP2) of the secondnucleotide sequence converts the blocked nucleic acid molecule to asingle strand unblocked nucleic acid molecule. The unblocked nucleicacid molecule contains only the first nucleotide sequence, which hassufficient complementarity to hybridize to the gRNA of RNP2, therebyactivating the trans-endonuclease activity of RNP2.

In some embodiments, the second nucleotide sequence at least partiallyhybridizes to the first nucleotide sequence, resulting in a secondarystructure containing at least one loop (e.g., hairpin loops, tetraloops,pseudoknots, junctions, kissing hairpins, internal loops, bulges, andmultibranch loops). Such loops block the nucleic acid molecule frombinding or incorporating into an RNP complex thereby initiating cis- ortrans-cleavage (see, e.g., the exemplary structures in FIGS. 2C-2E).

In some embodiments, the blocked nucleic acid molecule may contain aprotospacer adjacent motif (PAM) sequence, or partial PAM sequence,positioned between the first and second nucleotide sequences, where thefirst sequence is 5′ to the PAM sequence, or partial PAM sequence.Inclusion of a PAM sequence may increase the reaction kineticsinternalizing the unblocked nucleic acid molecule into RNP2 and thusdecrease the time to detection. In other embodiments, the blockednucleic acid molecule does not contain a PAM sequence.

In some embodiments, the blocked nucleic acid molecules (i.e., high IQnucleic acid molecules—in relation to binding to RNP2) of the disclosuremay include a structure represented by Formula I (e.g., FIG. 2C),Formula II (e.g., FIG. 2D), Formula III (e.g., FIG. 2E), or Formula N(e.g., FIG. 2F) wherein Formulas I-N are in the 5′-to-3′ direction:A-(B-L)_(J)-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;    -   B is 4-12 nucleotides in length;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10;    -   C is 4-15 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then A-(B-L)J-C and T-D are separate nucleic acid        strands;    -   T is 17-135 nucleotides in length (e.g., 17-100, 17-50, or        17-25) and comprises a sequence complementary to B and C; and    -   D is 0-10 nucleotides in length and comprises a sequence        complementary to A;        D-T-T′-C-(L-B)_(J)-A  (Formula II);    -   wherein D is 0-10 nucleotides in length;    -   T-T′ is 17-135 nucleotides in length (e.g., 17-100, 17-50, or        17-25);    -   T′ is 1-10 nucleotides in length and does not hybridize with T;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T;    -   L is 3-25 nucleotides in length and does not hybridize with T;    -   B is 4-12 nucleotides in length and comprises a sequence        complementary to T;    -   J is an integer between 1 and 10;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D;        T-D-M-A-(B-L)_(J)-C  (Formula III);    -   wherein T is 17-135 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-10 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then T-D and A-(B-L)_(J)-C are separate nucleic acid        strands;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D;    -   B is 4-12 nucleotides in length and comprises a sequence        complementary to T;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10; and    -   C is 4-15 nucleotides in length;        T-D-M-A-Lp-C  (Formula N);    -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-15 nucleotides in length;    -   M is 1-25 nucleotides in length;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D; and    -   L is 3-25 nucleotides in length;    -   p is 0 or 1;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T.        In alternative embodiments of any of these molecules, T (or        T-T′) can have a maximum length of 1000 nucleotides, e.g., at        most 750, at most 500, at most 250, at most 200, at most 135, at        most 75, at most 50, or at most 25.

Nucleotide mismatches can be introduced in any of the above structurescontaining double strand segments (for example, where M is absent inFormula I or Formula III) to reduce the melting temperature (Tm) of thesegment such that once the loop (L) is cleaved, the double strandsegment is unstable and dehybridizes rapidly. The percentage ofnucleotide mismatches of a given segment may vary between 0% and 50%;however, the maximum number of nucleotide mismatches is limited to anumber where the secondary loop structure still forms. “Segments” in theabove statement refers to A, B, and C. In other words, the number ofhybridized bases can be less than or equal to the length of each doublestrand segment and vary based on number of mismatches introduced.

In any blocked nucleic acid molecule having the structure of Formula I,III, or N, T will have sequence complementarity to a nucleotide sequence(e.g., a spacer sequence) within a gRNA of RNP2. The nucleotide sequenceof T is to be designed such that hybridization of T to the gRNA of RNP2activates the trans-nuclease activity of RNP2. In any blocked nucleicacid molecule having structure of Formula II, T-T′ will have sequencecomplementarity to a sequence (e.g., a spacer sequence) within the gRNAof RNP2. The nucleotide sequence of T-T′ is to be designed such thathybridization of T-T′ τo the gRNA of RNP2 activates the trans-cleavageactivity of RNP2. For T or T-T′, full complementarity to the gRNA is notnecessarily required, provided there is sufficient complementarity tocause hybridization and trans-cleavage activation of RNP2.

In any of the foregoing embodiments, the blocked nucleic acid moleculesof the disclosure may and preferably do further contain a reportermoiety attached thereto such that cleavage of the blocked nucleic acidreleases a signal from the reporter moiety. (See FIG. 4 , mechanismsdepicted at center and bottom.)

Also, in any of the foregoing embodiments, the blocked nucleic acidmolecule may be a modified or non-naturally occurring nucleic acidmolecule. In some embodiments, the blocked nucleic acid molecules of thedisclosure may further contain a locked nucleic acid (LNA), a bridgednucleic acid (BNA), and/or a peptide nucleic acid (PNA). The blockednucleic acid molecule may contain a modified or non-naturally occurringnucleoside, nucleotide, and/or internucleoside linkage, such as a2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modifiednucleoside, and a phosphorothioate (PS) bond, any other nucleic acidmolecule modifications described above, and any combination thereof.

The Signal Boosting Cascade Assay Employing Blocked Primer Molecules

The blocked nucleic acids described above may also, in an alternativeembodiment, be blocked primer molecules. Blocked primer moleculesinclude a sequence complementary to a primer binding domain (PBD) on atemplate molecule (see description below in reference to FIGS. 3A and3B) and can have the same general structures as the blocked nucleic acidmolecules described above. A PBD serves as a nucleotide sequence forprimer hybridization followed by primer extension by a polymerase. Inany of Formulas I, II, or III described above, the blocked primernucleic acid molecule may include a sequence complementary to the PBD onthe 5′ end of T. The unblocked primer nucleic acid molecule can bind toa template molecule at the PBD and copy the template molecule viapolymerization by a polymerase.

Other specific embodiments of the cascade assay that utilize blockedprimer molecules are depicted in FIGS. 3A and 3B. In the embodimentsusing blocked nucleic acid molecules described above, activation of RNP1and trans-cleavage of the blocked nucleic acid molecules were used toactivate RNP2—that is, the unblocked nucleic acid molecules are a targetsequence for the gRNA in RNP2. In contrast in the embodiments usingblocked primers, activation of RNP1 and trans-cleavage unblocks ablocked primer molecule that is then used to prime a template moleculefor extension by a polymerase, thereby synthesizing activating moleculesthat are the target sequence for the gRNA in RNP2.

FIG. 3A is a diagram showing the sequence of steps in an exemplarycascade assay (300) involving circular blocked primer molecules andlinear template molecules. At left of FIG. 3A is a cascade assayreaction mixture comprising 1) RNP1 s (301) (only one RNP1 is shown); 2)RNP2s (302); 3) linear template molecules (330) (which is the non-targetstrand); 4) a circular blocked primer molecule (334) (i.e., a high K_(d)molecule); and 5) a polymerase (338), such as a Phi29 (029) polymerase.The linear template molecule (330) (non-target strand) comprises a PAMsequence (331), a primer binding domain (PBD) (332) and, optionally, anucleoside modification (333) to protect the linear template molecule(330) from 3′→5′ exonuclease activity. Blocked primer molecule (334)comprises a cleavable region (335) and a complementary region (336) tothe PBD (332) on the linear template molecule (330).

Upon addition of a sample comprising a target nucleic acid of interest(304) (capable of complexing with the gRNA in RNP1 (301)), the targetnucleic acid of interest (304) combines with and activates RNP1 (305)but does not interact with or activate RNP2 (302). Once activated, RNP1cuts the target nucleic acid of interest (304) via sequence-specificcis-cleavage, which activates non-specific trans-cleavage of othernucleic acids present in the reaction mixture, including at least one ofthe blocked primer molecules (334). The circular blocked primer molecule(334) (i.e., a high K_(d) molecule, where high K_(d) relates to bindingto RNP2) upon cleavage becomes an unblocked linear primer molecule (344)(a low K_(d) molecule, where low K_(d) relates to binding to RNP2),which has a region (336) complementary to the PBD (332) on the lineartemplate molecule (330) and can bind to the linear template molecule(330).

Once the unblocked linear primer molecule (344) and the linear templatemolecule (330) are hybridized (i.e., hybridized at the PBD (332) of thelinear template molecule (330) and the PBD complement (336) on theunblocked linear primer molecule (344)), 3′→5′ exonuclease activity ofthe polymerase (338) removes any unhybridized single-stranded DNA at theend of the unblocked primer molecule (344) and the polymerase (338) cancopy the linear template molecule (330) to produce a synthesizedactivating molecule (346) which is a complement of the non-targetstrand, which is a target strand. The synthesized activating molecule(346) is capable of binding to the gRNA (306) of RNP2 and activatingRNP2 (302→308). As described above, because the nucleic acid-guidednuclease in the RNP2 (308) complex exhibits (that is, possesses) bothcis- and trans-cleavage activity, more blocked primer molecules (334)become unblocked primer molecules (344) triggering activation of moreRNP2s (308) and more trans-cleavage activity in a cascade. As statedabove in relation to blocked and unblocked nucleic acid molecules (bothlinear and circular), the unblocked primer molecule has a higher bindingaffinity for the gRNA in RNP2 than does the synthesized activatingmolecule, although there may be some “leakiness” where some blockedprimer molecules are able to interact with the gRNA in RNP2. However, anunblocked primer molecule has a substantially higher likelihood than ablocked primer molecule to hybridize with the gRNA of RNP2.

FIG. 3A at bottom depicts the concurrent activation of reportermoieties. Intact reporter moieties (309) comprise a quencher (310) and afluorophore (311). As described above in relation to FIG. 1C, thereporter moieties are also subject to trans-cleavage by activated RNP1(305) and RNP2 (308). The intact reporter moieties (309) becomeactivated reporter moieties (312) when the quencher (310) is separatedfrom the fluorophore (311), and the fluorophore emits a fluorescentsignal (313). Signal strength increases rapidly as more blocked primermolecules (334) become unblocked primer molecules (344) generatingsynthesized activating molecules (346) and triggering activation of moreRNP2 (308) complexes and more trans-cleavage activity of the reportermoieties (309). Again, here the reporter moieties are shown as separatemolecules from the blocked nucleic acid molecules, but otherconfigurations may be employed and are discussed in relation to FIG. 4 .Also, as with the cascade assay embodiment utilizing blocked nucleicacid molecules that are not blocked primers, with the exception of thegRNA in RNP1, the cascade assay components may stay the same no matterwhat target nucleic acid(s) of interest are being detected. Further, thecascade assay is tunable by employing blocked primer molecules havingdifferent configurations (i.e., loop sizes, clamp sizes, GC content) andthus different free energies.

FIG. 3B is a diagram showing the sequence of steps in an exemplarycascade assay (350) involving blocked primer molecules and circulartemplate molecules. The cascade assay of FIG. 3B differs from thatdepicted in FIG. 3A by the configuration of the template molecule. Wherethe template molecule in FIG. 3A was linear, in FIG. 3B the templatemolecule is circular. At left in FIG. 3B is a cascade assay reactionmixture comprising 1) RNP1s (301) (only one RNP1 is shown); 2) RNP2s(302); 3) a circular template molecule (352) (non-target strand); 4) acircular blocked primer molecule (334); and 5) a polymerase (338), suchas a X29 polymerase. The circular template molecule (352) (non-targetstrand) comprises a PAM sequence (331) and a primer binding domain (PBD)(332). Blocked primer molecule (334) comprises a cleavable region (335)and a complementary region (336) to the PBD (332) on the circulartemplate molecule (352).

Upon addition of a sample comprising a target nucleic acid of interest(304) (capable of complexing with the gRNA in RNP1 (301)), the targetnucleic acid of interest (304) combines with and activates RNP1 (305)but does not interact with or activate RNP2 (302). Once activated, RNP1cuts the target nucleic acid of interest (304) via sequence-specificcis-cleavage, which activates non-specific trans-cleavage of othernucleic acids present in the reaction mixture, including at least one ofthe blocked primer molecules (334). The circular blocked primer molecule(334), upon cleavage, becomes an unblocked linear primer molecule (344),which has a region (336) complementary to the PBD (332) on the circulartemplate molecule (352) and can hybridize with the circular templatemolecule (352).

Once the unblocked linear primer molecule (344) and the circulartemplate molecule (352) are hybridized (i.e., hybridized at the PBD(332) of the circular template molecule (352) and the PBD complement(336) on the unblocked linear primer molecule (344)), 3′→5′ exonucleaseactivity of the polymerase (338) removes any unhybridizedsingle-stranded DNA at the 3′ end of the unblocked primer molecule(344). The polymerase (338) can now use the circular template molecule(352) (non-target strand) to produce concatenated activating nucleicacid molecules (360) (which are concatenated target strands), which willbe cleaved by the trans-cleavage activity of activated RNP1. The cleavedregions of the concatenated synthesized activating molecules (360)(target strand) are capable of binding to the gRNA (306) of RNP2 andactivating the RNP2 (302 308) complex.

As described above, because the nucleic acid-guided nuclease in RNP2(308) comprises both cis- and trans-cleavage activity, more blockedprimer molecules (334) become unblocked primer molecules (344)triggering activation of more RNP2s (308) and more trans-cleavageactivity in a cascade. FIG. 3B at bottom depicts the concurrentactivation of reporter moieties. Intact reporter moieties (309) comprisea quencher (310) and a fluorophore (311). As described above in relationto FIG. 1C, the reporter moieties are also subject to trans-cleavage byactivated RNP1 (305) and RNP2 (308). The intact reporter moieties (309)become activated reporter moieties (312) when the quencher (310) isseparated from the fluorophore (311), and the fluorescent signal (313)is unquenched and can be detected. Signal strength increases rapidly asmore blocked primer molecules (334) become unblocked primer molecules(344) generating synthesized activating nucleic acid molecules andtriggering activation of more RNP2s (308) and more trans-cleavageactivity of the reporter moieties (309). Again, here the reportermoieties are shown as separate molecules from the blocked nucleic acidmolecules, but other configurations may be employed and are discussed inrelation to FIG. 4 . Also note that as with the other embodiments of thecascade assay, in this embodiment, with the exception of the gRNA inRNP1, the cascade assay components can stay the same no matter whattarget nucleic acid(s) of interest are being detected.

The polymerases used in the “blocked primer molecule” embodiments serveto polymerize a reverse complement strand of the template molecule(non-target strand) to generate a synthesized activating molecule(target strand) as described above. In some embodiments, the polymeraseis a DNA polymerase, such as a BST, T4, or Therminator polymerase (NewEngland BioLabs Inc., Ipswich MA., USA). In some embodiments, thepolymerase is a Klenow fragment of a DNA polymerase. In some embodimentsthe polymerase is a DNA polymerase with 5′→3′ DNA polymerase activityand 3‘-’ 5′ exonuclease activity, such as a Type I, Type II, or Type IIIDNA polymerase. In some embodiments, the DNA polymerase, including theX29, T7, Q5®, Q5U®, Phusion®, OneTaq®, LongAmp®, Vent®, or Deep Vent®DNA polymerases (New England BioLabs Inc., Ipswich MA., USA), or anyactive portion or variant thereof. Also, a 3′ to 5′ exonuclease can beseparately used if the polymerase lacks this activity.

FIG. 4 depicts three mechanisms in which a cascade assay reaction canrelease a signal from a reporter moiety. FIG. 4 at top shows themechanism discussed in relation to FIGS. 2A, 3A and 3B. In thisembodiment, a reporter moiety (409) is a separate molecule from theblocked nucleic acid molecules present in the reaction mixture. Reportermoiety (409) comprises a quencher (410) and a fluorophore (411). Anactivated reporter moiety (412) emits a signal from the fluorophore(411) once it has been physically separated from the quencher (410).Again, if the reporter moiety is a separate molecule that is notactivated as part of the blocked nucleic acid molecule (or blockedprimer molecule), then activation kinetics of the reporter will be morerapid; however, if activation of the reporter moiety is coupled tounblocking of the blocked nucleic acid molecules (or blocked primermolecules), activation kinetics will be slower.

FIG. 4 at center shows a blocked nucleic acid molecule (403), which isalso a reporter moiety. In addition to quencher (410) and fluorophore(411), a blocking moiety (407) can be seen (see also blocked nucleicacid molecules 203 in FIG. 2A). Blocked nucleic acid molecule/reportermoiety (403) comprises a quencher (410) and a fluorophore (411). In thisembodiment of the cascade assay, when the blocked nucleic acid molecule(403) is unblocked due to trans-cleavage initiated by the target nucleicacid of interest binding to RNP1, the unblocked nucleic acid molecule(406) also becomes an activated reporter moiety with fluorophore (411)separated from quencher (410). Note both the blocking moiety (407) andthe quencher (410) are removed. In this embodiment, reporter signal isdirectly generated as the blocked nucleic acid molecules becomeunblocked.

FIG. 4 at the bottom shows that cis-cleavage of an unblocked nucleicacid or a synthesized activation molecule at a PAM distal sequence byRNP2 generates a signal. Shown are activated RNP2 (408), unblockednucleic acid molecule (461), quencher (410), and fluorophore (411)forming an activated RNP2 with the unblocked nucleic acid/reportermoiety intact (460). Cis-cleavage of the unblocked nucleic acid/reportermoiety (461) results in an activated RNP2 with the reporter moietyactivated (462), comprising the activated RNP2 (408), the unblockednucleic acid molecule with the reporter moiety activated (463), quencher(410) and fluorophore (411).

Tuning the Cascade Assay Using Blocked Nucleic Acid Molecules or BlockedPrimer Molecules

The present disclosure improves upon the signal cascade assay describedin U.S. Ser. Nos. 17/861,207; 17/861,208; and 17/861,209 by configuringthe blocked nucleic acid molecules or blocked primer molecules toincrease reaction kinetics, decrease reaction kinetics, providedetection over a large range of concentrations of the target nucleicacids of interest or provide accurate quantification of target nucleicacids of interest within a narrow range of concentrations. As describedabove in detail in relation to FIGS. 1C, 2A, 2B, 3A, 3B, and 4 , thecascade assay is initiated when a target nucleic acid of interest bindsto and activates a first pre-assembled ribonucleoprotein complex (RNP1).The guide nucleic acid of RNP1 (i.e., gRNA1), comprising a sequencecomplementary to the target nucleic acid of interest, guides RNP1 to thetarget nucleic acid of interest. Upon binding of N nucleotide bases ofthe target nucleic acid of interest to RNP1, RNP1 becomes activated,cleaving the target nucleic acid of interest in a sequence-specificmanner (i.e., cis-cleavage) leading to non-sequence-specific,indiscriminate trans-cleavage activity which unblocks the blockednucleic acid molecules in the reaction mixture. The unblocked nucleicacid molecules can then activate a second pre-assembledribonucleoprotein complex (RNP2), where RNP2 comprises a second gRNA(gRNA2) comprising a sequence complementary to the unblocked nucleicacid molecules, and at least one of the unblocked nucleic acid moleculesis cleaved in a sequence-specific manner. Cis-cleavage of the unblockednucleic acid molecule then leads to non-sequence-specific,indiscriminate trans-cleavage activity by RNP2, which in turn unblocksmore blocked nucleic acid molecules (and reporter moieties) in thereaction mixture activating more RNP2s. Each newly activated RNP2activates more RNP2s, which in turn cleave more blocked nucleic acidmolecules and reporter moieties in a reaction cascade.

The improvement to the signal cascade or signal boost cascade assaydescribed herein is drawn to being able to “tune” the cascade assay byemploying differently configured blocked nucleic acid molecules (orblocked primer molecules) that activate RNP2. “Tuning” relates tocontrolling kinetics of the assay by two orders of magnitude, from underone minute of target nucleic acids of interest to detection over 100minutes or more. The present disclosure demonstrates that by alteringthe Gibbs free energy (i.e., molecular configuration and composition)via varying loop numbers, “clamp” lengths, and GC content of the blockednucleic acid molecules employed in the cascade assay, the kinetics ofthe cascade assay can be “tuned” regardless of RNP1 targetconcentrations.

There are various methods to calculate free energy (i.e., Gibbs freeenergy). In one method, Gibbs free energy changes can be calculatedusing enthalpy and entropy values according to:ΔG°(T)=(ΔH°−TΔS°)cal mol⁻¹where T is the temperature at which Gibbs free energy is assessed.Hybridization enthalpy (ΔH°) was calculated as a difference of theoligonucleotides' total energy in the double-stranded (ds) states(E^(ds) _(tot)) and in the single-stranded (ss) states (E^(ss1) _(tot)and E^(ss2) _(tot)):ΔH°≈E ^(ds) _(tot)−(E ^(ss1) +E ^(ss2))There is a linear dependence of the hybridization entropy on theenthalpy of the complex formation with a very high correlationcoefficient (R2=0.995). This dependence is described by the equation:ΔS°=2.678ΔH°/1000−6.0 cal mol⁻¹ K−1.(See, e.g., Lomzov, et al., J. Phys. Chem., 119(49):15221-234 (°15).

In another method, Gibbs free energy is calculated using enthalpy andentropy values. The following formula is used to calculate the freeenergy for each base pair:ΔG° (T)=(ΔH°−TΔS°)cal mol⁻¹The total ΔG° is given by:ΔG° (total)=Σ_(i) n _(i) ΔG° (i)+ΔG° (init with term G·C)+ΔG° (init withterm A·T)+ΔG° (sym)Where ΔG° (i) are the standard free energy changes for the 10 possibleWatson-Crick NNs (e.g., ΔG° (1)=ΔG°₃₇ (AA/TT), ΔG° (2)=ΔG°₃₇ (TA/AT), .. . etc.), n; is the number of occurrences of each nearest neighbor, i,and ΔG° (sym) equals +0.43 kcal/mol (1 cal=4.184 J) if the duplex isself-complementary and zero if it is non-self-complementary.

An example of total Gibbs free energy is shown on CGTTGA·TCAACGhybridized DNA:

    ↓   ↓   ↓ 5′ C-G-T-T-G-A 3′    * * * * * * 3′ G-C-A-A-C-T 5′      ↑   ↑ΔG° ₃₇(pred)=ΔG° ₃₇(CG/GC)+ΔG°37(GT/CA)+ΔG° ₃₇(TT/AA)+ΔG° ₃₇(TG/AC)+ΔG°₃₇(GA/CT)+ΔG°(init.)=−2.17-1.44-1.00-1.45-1.30+0.98+1.03ΔG° ₃₇(pred.)=−5.35 kcal/molΔG° ₃₇(obs.)=−5.20 kcal/mol

The ΔH° and ΔS° parameters are analogously calculated from theparameters in Table 1.

TABLE 1 Unified oligonucleotides ΔHº and  ΔSº NN parameters in 1 M NaClSequence ΔHº kcal/mol ΔSº kcal/mol AA/TT  -7.9 -22.2 AT/TA  -7.2 -20.4TA/TA  -7.2 -21.3 CA/GT  -8.5 -22.7 GT/CA  -8.4 -22.4 CT/GA  -7.8 -21.0GA/CT  -8.2 -22.2 CG/GC -10.6 -27.2 GC/CG  -9.8 -24.4 GG/CC  -8.0 -19.9Init. w/ term. G-C   0.1  -2.8 Init. w/ term. A-T   2.3   4.1Symmetry correction   0  -1.4See, e.g., SantaLucia, et al., PNAS, 95(4):1460-65 (1998).

Long regions of hybridization (or self-hybridization), i.e., the“clamps” or “clamp regions” of the blocked nucleic acid molecules, leadto higher T_(m) and thus slower kinetics. Further, the more loopspresent that need to be cleaved to unblock the blocked nucleic acidmolecules or blocked primer molecules, the slower the reaction kineticswill be. Also, if the reporter moiety is incorporated into the blockednucleic acid molecule design, the detection kinetics of the reportermoiety will be slow compared to when reporter moieties are present inthe cascade assay reaction mixture as separate molecules. Finally, withthe blocked nucleic acid molecules and the blocked primer molecules, thereaction rate increases as GC content increases and the reaction ratedecreases as GC content decreases, particularly in relation to the GCcontent of a clamp region. Reaction kinetics of course are also affectedby temperature. The higher the temperature, the more rapid the reaction.

Like Gibbs free energy, melting temperature (T_(m)) can be calculatedusing one of several calculations known in the art. For example, forsequences less than 14 nucleotides, the formula is:Tm=(wA+xT)*2+(yG+zC)*4

-   -   where w, x, y, z are the number of bases A, T, G, C in the        sequence respectively. For sequences longer than 13 nucleotides,        the equation used is:        Tm=64.9+41*(yG+zC−16.4)/(wA+xT+yG+zC)        Both equations assume that annealing occurs under the standard        conditions of 50 nM primer, 50 nM Na⁺, and pH 7.0. See, e.g.,        Mamur and Doty, JMB, 5(1):109-18 (1962) and Wallace, et al.,        NAR, 6:3543-57 (1979).

In distinguishing blocked nucleic acid molecules, the Gibbs free energyof the blocked nucleic acid molecules (or blocked primer molecules) hasto be negative enough to be stable in the cascade assay reaction mixtureunder the desired assay conditions. If the blocked nucleic acidmolecules are not stable, unblocking will not be specific; that is,unblocking the blocked nucleic acid molecules may take place withoutactivation of RNP1 by the target nucleic acid of interest, resulting infalse positive. For example, note that the clamp regions of molecule U29(FIG. 6A) are 5 and 6 basepairs in length, resulting in a blockednucleic acid molecule with a Gibbs free energy of −5.85 kcal/mol.Shorter clamps of, e.g., 4 basepairs in length may result in an unstableblocked nucleic acid molecule at the reaction temperature of 25° C. (seethe assay results shown in FIGS. 6B-6H and the descriptions thereofbelow) and thus would be unsuitable for the cascade assay reaction evenif instantaneous detection is desired. Thus, at a reaction temperatureof 25° C., the Gibbs free energy of the blocked nucleic acid moleculewill be about −5.5 kcal/mol to about −20.0 kcal/mol, or about −6.0kcal/mol to about −18.0 kcal/mol, or about −8.0 kcal/mol to about −16.0kcal/mol. If faster kinetics are desired, at a reaction temperature of25° C., the Gibbs free energy of the blocked nucleic acid molecule willbe about −5.0 kcal/mol to about −12.0 kcal/mol, or about −6.0 kcal/molto about 10.0 kcal/mol. If faster kinetics are desired, at a reactiontemperature of 25° C., the Gibbs free energy of the blocked nucleic acidmolecule will be about −12.0 kcal/mol to about −20.0 kcal/mol, or about−14.0 kcal/mol to about −18.0 kcal/mol. In addition, the tunable blockednucleic acid molecules can comprise a PAM sequence or lack a PAMsequence, but if a PAM sequence is present, it is present in a loopsequence.

In addition to slowing down the cascade assay reaction kinetics in acustomizable manner, increasing reaction kinetics (i.e., slower=morequantifiable) allows for quantification of small differences in thenumber of target nucleic acids of interest, in an almost digital manner.For example, the configuration of the blocked nucleic acid molecule canbe chosen so as to, e.g., distinguish between one copy of a targetnucleic acid of interest from two copies of the target nucleic acid ofinterest, or, e.g., two copies of a target nucleic acid of interest fromthree copies of the target nucleic acid of interest, or e.g., threecopies of a target nucleic acid of interest from five copies of thetarget nucleic acid of interest, or e.g., ten copies of a target nucleicacid of interest from fifteen copies of the target nucleic acid ofinterest. Again, the higher the Tm of a blocked nucleic acid molecule orblocked primer molecule, the more loops that need to be cleaved, thelonger the clamp regions, the higher the GC content of the claimregions, the slower the reaction kinetics leading to more distinctionbetween small differences in copy number. Once putative blocked nucleicacid molecules or blocked primer molecules are designed, Gibbs freeenergy is calculated and then a selection of blocked nucleic acidmolecules or blocked primer molecules are tested under various reactionconditions. The choice of a final blocked nucleic acid molecule orblocked primer molecule candidate is thus determined empirically.

Selecting the optimal design of the blocked nucleic acid molecule (orblocked primer molecule) for detection of a specific target nucleic acidof interest in a desired range of copy numbers requires selection of adesired reaction temperature and experimentation as described below toestablish detection curves as shown in FIGS. 6B-6H, 7B, 8B, 9B, 10B and11 b. Once a “sweet spot” for copy number detection (and reaction rate)is achieved, the cascade assay can be programmed to detect virtually anytarget nucleic acid of interest (or combinations thereof) by changingthe guide nucleic acid(s) in RNP1. The cascade assay reaction mayproceed to completion with measurement continuously or at specifictimepoints, and/or the cascade assay reaction may be arrested orquenched at a desired timepoint by, e.g., addition of EDTA.

Applications of the Cascade Assay

The present disclosure describes cascade assays for detecting one ormore target nucleic acids of interest in a sample. The cascade assaysallow for massive multiplexing and minimum workflow yet provide accurateresults at low cost. In embodiments, the cascade assay can be tuned todetect target nucleic acids of interest instantaneously or nearly so,even at ambient temperatures above 16° C.; detect target nucleic acidsof interest over a longer period of time; detect target nucleic acids ofinterest over large copy number concentrations; or detect copies oftarget nucleic acids of interest quantitatively over a small range in analmost digital manner. That is, the present disclosure describes methodsto “tune” the assay such that reaction kinetics can be controlled overmultiple orders of magnitude. Moreover, the various embodiments of thecascade assay are notable in that, with the exception of the gRNA inRNP1, the cascade assay components can stay the same no matter whattarget nucleic acid(s) of interest are being detected and RNP1 is easilyreprogrammed. Further, this remains true in the context of tunability,as the cascade assay is tunable by use of different blocked nucleic acidmolecules (or blocked primer molecules) used to activate RNP2 oncedesired reaction kinetics have been chosen and is independent of theRNP1 target nucleic acid concentration. Note this is not true of, e.g.,PCR, where the Ct value depends on the concentration of the targetnucleic acid.

If single copy differences in the number of target nucleic acids ofinterest are required, such as, e.g., in oncology applications, one candesign the best blocked nucleic acid molecule for this purpose. Such ablocked nucleic acid molecule might comprise a Gibbs free energy of −15to −20 kcal/mol, such as molecules T135, T134, or T119 seen in FIGS. 9A,10A and 11A, respectively. If, in contrast, determining the presence ofa target nucleic acid of interest virtually instantaneously is desired,again the best blocked nucleic acid for this purpose can be designed,and may comprise a Gibbs free energy of approximately −5 kcal/mol and amolecular structure similar to, e.g., that of U29 seen in FIG. 6A.

Target nucleic acids of interest are derived from samples as describedin more detail above. Suitable samples for testing include, but are notlimited to, any environmental sample, such as air, water, soil, surface,food, clinical sites and products, industrial sites and products,pharmaceuticals, medical devices, nutraceuticals, cosmetics, personalcare products, agricultural equipment and sites, and commercial samples,and any biological sample obtained from an organism or a part thereof,such as a plant, animal, or microbe. In some embodiments, the biologicalsample is obtained from an animal subject, such as a human subject. Abiological sample is any solid or fluid sample obtained from, excretedby or secreted by any living organism, including, without limitation,single celled organisms, such as bacteria, yeast, protozoans, andamoebas among others, multicellular organisms including plants oranimals, including samples from a healthy or apparently healthy humansubject or a human patient affected by a condition or disease to bediagnosed or investigated, such as an infection with a pathogenicmicroorganism, such as a pathogenic bacteria or virus.

For example, a biological sample can be a biological fluid obtained froma human or non-human (e.g., livestock, pets, wildlife) animal, and mayinclude but is not limited to blood, plasma, serum, urine, stool,sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleuraleffusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreoushumor, or any bodily secretion, a transudate, an exudate (for example,fluid obtained from an abscess or any other site of infection orinflammation), or fluid obtained from a joint (for example, a normaljoint or a joint affected by disease, such as rheumatoid arthritis,osteoarthritis, gout or septic arthritis), or a swab of skin or mucosalmembrane surface (e.g., a nasal or buccal swab).

In some embodiments, the sample can be a viral or bacterial sample or abiological sample that has been minimally processed, e.g., only treatedwith a brief lysis step prior to detection. In other embodiments,minimal processing can include thermal lysis at an elevated temperatureto release nucleic acids. Suitable methods are contemplated in U.S. Pat.No. 9,493,736, among other references. Common methods for cell lysisinvolve thermal, chemical, enzymatic, or mechanical treatment of thesample or a combination of those (see, e.g., Example I below). In someembodiments, minimal processing can include treating the sample withchaotropic salts such as guanidine isothiocyanate or guanidine HCl.Suitable methods are contemplated in U.S. Pat. Nos. 8,809,519 and7,893,251, among other references. In some embodiments, minimalprocessing may include contacting the sample with reducing agents suchas DTT or TCEP and EDTA to inactivate inhibitors and/or other nucleasespresent in the crude samples. In other embodiments, minimal processingfor biofluids may include centrifuging the samples to obtain cell-debrisfree supernatant before applying the reagents. Suitable methods arecontemplated in U.S. Pat. No. 8,809,519, among other references. Instill other embodiments, minimal processing may include performingDNA/RNA extraction to get purified nucleic acids before applying CRISPRCascade reagents.

Table 2 below lists exemplary commercial sample processing kits, andTable 3 below lists point of care processing techniques.

TABLE 2 Exemplary Commercial Sample and Nucleic Acid Processing KitsManufacturer Kit Sample Type Output Lysing and extraction methodsQiagen ® DNeasy ™ Blood small volumes genomic Isolation of Genomic DNAfrom & Tissue Kits of blood DNA Small Volumes of Blood dried blood 1.Uses Chemical and spots Biological/Enzymatic lysis methods urine 2. Usessolid phase extraction (SPE) tissues with Column Purification laser-Isolation of Genomic DNA from microdissected Tissues tissues 1. UsesChemical and Biological/Enzymatic lysis methods 2. Used to dissolve andlyse tissue sections completely, higher temperature and longer timeincubations up to 24 hours are used Qiagen ® QIAamp ® UCP whole bloodmicrobial Specific pretreatment protocols are Pathogen swabs DNAsuggested depending on sample type Mini Handbook cultures - with orwithout the use of kits for microbial DNA pelleted Mechanical LysisMethod before purification microbial cells downstream applications. bodyfluids Downstream applications contain: 1. Chemical andBiological/Enzymatic lysis methods 2. SPE with Column PurificationQiagen ® QIAamp ® Viral plasma and viral DNA 1. Uses Chemical lysismethods RNA Kits serum 2. Uses SPE with Column CSF Purification urineother cell-free body fluids cell-culture supernatants swabs Zymo Quick-whole blood genomic 1. Uses chemical lysis methods Research ™DNA ™Microprep plasma DNA 2. Uses SPE with column Kit serum purificationbody fluids buffy coat lymphocytes swabs cultured cells Zymo Quick-DNA ™A. fumigatus Microbial Uses Bead lysis and pretreatment Research ™Fungal/Bacterial C. albicans DNA with: Miniprep Kit N. crassa 1.Chemical lysis methods with S. cerevisiae chaotropic salts S. pombe 2.Nucleic acid extraction (NAE) mycelium with SPE with silica matricesGram positive bacteria Gram negative bacteria

TABLE 3 Point of Care Sample Processing Techniques Steps ProtocolExample 1 Protocol Example 2 Protocol Example 3 Field-deployable viralStreamlined Lucira Health ™ diagnostics using inactivation, CRISPR-Cas13amplification, and Science, Cas13-based detection 27; 360(6387): 444-448of SARS-CoV-2 (2018) Nat Commun, 11: 5921 (2020) 1. Cell disruptionSamples were thermally A nasopharyngeal (NP) Lucira Health uses a(lysis) and treated at ~40° C. for ~15 swab or saliva sample singlebuffer that lyses inactivation of minutes for nuclease was lysed and andinactivates nucleases deactivation, thereafter inactivated for 10nucleases and/or In point-of-care setting, at 90° C. for 5 minutesminutes with thermal inhibitors. cell disruption and for viraldeactivation. treatment. These A nasal swab is directly inactivation ofSample Types: samples were incubated added to a single nucleases is doneUrine for 5 min at 40° C., lysing/reaction buffer commonly throughSaliva followed by 5 min at and vigorously stirred thermal lysis.Diluted blood 70° C. (or 5 min at 95° C., to release the viral (1:3 withPBS) if saliva) particulates from the Targets: Viruses swab. Target:SARS-Cov-2 2. Assay on crude Thermally treated Thermally treatedProcessed biological sample biological samples biological samples sampleis used in an This is usually a direct (above) were used (above) wereused isothermal reaction for assay on the crude directly for directlyfor pathogenic nucleic acid sample post cell amplification andamplification and detection. disruption and detection of pathogenicdetection of pathogenic inactivation of nucleic acid. nucleic acid.nucleases. No extraction is usually performed.

FIG. 5 shows a lateral flow assay (LFA) device that can be used todetect the cleavage and separation of a signal from a reporter moiety.For example, the reporter moiety may be a single-stranded ordouble-stranded oligonucleotide with terminal biotin and fluoresceinamidite (FAM) modifications; and, as described above, the reportermoiety may also be part of a blocked nucleic acid. The LFA device mayinclude a pad with binding particles, such as gold nanoparticlesfunctionalized with anti-FAM antibodies; a control line with a firstbinding moiety attached, such as avidin or streptavidin; a test linewith a second binding moiety attached, such as antibodies; and anabsorption pad. After completion of a cascade assay (see FIGS. 2A, 3A,and 3B), the assay reaction mixture is added to the pad containing thebinding particles, (e.g., antibody labeled gold nanoparticles). When thetarget nucleic acid of interest is present, a reporter moiety iscleaved, and when the target nucleic acid of interest is absent, thereporter is not cleaved.

A moiety on the reporter binds to the binding particles and istransported to the control line. When the target nucleic acid ofinterest is absent, the reporter moiety is not cleaved, and the firstbinding moiety binds to the reporter moiety, with the binding particlesattached. When the target nucleic acid of interest is present, oneportion of the cleaved reporter moiety binds to the first bindingmoiety, and another portion of the cleaved reporter moiety bound to thebinding particles via the moiety binds to the second binding moiety. Inone example, anti-FAM gold nanoparticles bind to a FAM terminus of areporter moiety and flow sequentially toward the control line and thento the test line. For reporters that are not trans-cleaved, goldnanoparticles attach to the control line via biotin-streptavidin andresult in a dark control line. In a negative test, since the reporterhas not been cleaved, all gold conjugates are trapped on control linedue to attachment via biotin-streptavidin. A negative test will resultin a dark control line with a blank test line. In a positive test,reporter moieties have been trans-cleaved by the cascade assay, therebyseparating the biotin terminus from the FAM terminus. For cleavedreporter moieties, nanoparticles are captured at the test line due toanti-FAM antibodies. This positive test results in a dark test line inaddition to a dark control line.

The components of the cascade assay may be provided in various kits fortesting at, e.g., point of care facilities, in the field, pandemictesting sites, and the like. In one aspect, the kit for detecting atarget nucleic acid of interest in a sample includes: firstribonucleoprotein complexes (RNP1s), second ribonucleoprotein complexes(RNP2s), blocked nucleic acid molecules, and reporter moieties. Thefirst complex (RNP1) comprises a first nucleic acid-guided nuclease anda first gRNA, where the first gRNA includes a sequence complementary tothe target nucleic acid(s) of interest. Binding of the first complex(RNP1) to the target nucleic acid(s) of interest activatestrans-cleavage activity of the first nucleic acid-guided nuclease. Thesecond complex (RNP2) comprises a second nucleic acid-guided nucleaseand a second gRNA that is not complementary to the target nucleic acidof interest. The blocked nucleic acid molecule comprises a sequencecomplementary to the second gRNA, where trans-cleavage of the blockednucleic acid molecule results in an unblocked nucleic acid molecule andthe unblocked nucleic acid molecule can bind to the second complex(RNP2), thereby activating the trans-cleavage activity of the secondnucleic acid-guided nuclease. Activating trans-cleavage activity in RNP2results in an exponential increase in unblocked nucleic acid moleculesand in active reporter moieties, where reporter moieties are nucleicacid molecules and/or are operably linked to the blocked nucleic acidmolecules and produce a detectable signal upon cleavage by RNP2.

In a second aspect, the kit for detecting a target nucleic acid moleculein sample includes: first ribonucleoprotein complexes (RNP1s), secondribonucleoprotein complexes (RNP2s), template molecules, blocked primermolecules, a polymerase, nucleotide triphosphates (NTPs), and reportermoieties. The first ribonucleoprotein complex (RNP1) comprises a firstnucleic acid-guided nuclease and a first gRNA, where the first gRNAincludes a sequence complementary to the target nucleic acid of interestand where binding of RNP1 to the target nucleic acid(s) of interestactivates trans-cleavage activity of the first nucleic acid-guidednuclease. The second complex (RNP2) comprises a second nucleicacid-guided nuclease and a second gRNA that is not complementary to thetarget nucleic acid of interest. The template molecules comprise aprimer binding domain (PBD) sequence as well as a sequence correspondingto a spacer sequence of the second gRNA. The blocked primer moleculescomprise a sequence that is complementary to the PBD on the templatenucleic acid molecule and a blocking moiety.

Upon binding to the target nucleic acid of interest, RNP1 becomes activetriggering trans-cleavage activity that cuts at least one of the blockedprimer molecules to produce at least one unblocked primer molecule. Theunblocked primer molecule hybridizes to the PBD of one of the templatenucleic acid molecules, is trimmed of any excess nucleotides by the3′-to-5′ exonuclease activity of the polymerase and is then extended bythe polymerase with NTPs to form a synthesized activating molecule witha sequence that is complementary to the second gRNA of RNP2. Uponactivating RNP2, additional trans-cleavage activity is initiated,cleaving at least one additional blocked primer molecule. Continuedcleavage of blocked primer molecules and subsequent activation of moreRNP2s proceeds at an exponential rate. A signal is generated uponcleavage of a reporter molecule by active RNP2 complexes; therefore, achange in signal production indicates the presence of the target nucleicacid molecule.

Any of the kits described herein may further include a sample collectiondevice, e.g., a syringe, lancet, nasal swab, or buccal swab forcollecting a biological sample from a subject, and/or a samplepreparation reagent, e.g., a lysis reagent. Each component of the kitmay be in separate container or two or more components may be in thesame container. The kit may further include a lateral flow device usedfor contacting the biological sample with the reaction mixture, where asignal is generated to indicate the presence or absence of the targetnucleic acid molecule of interest. In addition, the kit may furtherinclude instructions for use and other information.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example I: Preparation of Nucleic Acids of Interest

Mechanical lysis: Nucleic acids of interest may be isolated by variousmethods depending on the cell type and source (e.g., tissue, blood,saliva, environmental sample, etc.). Mechanical lysis is a widely-usedcell lysis method and may be used to extract nucleic acids frombacterial, yeast, plant and mammalian cells. Cells are disrupted byagitating a cell suspension with “beads” at high speeds (beads fordisrupting various types of cells can be sourced from, e.g., OPSDiagnostics (Lebanon NJ, US) and MP Biomedicals (Irvine, CA, USA)).Mechanical lysis via beads begins with harvesting cells in a tissue orliquid, where the cells are first centrifuged and pelleted. Thesupernatant is removed and replaced with a buffer containing detergentsas well as lysozyme and protease. The cell suspension is mixed topromote breakdown of the proteins in the cells and the cell suspensionthen is combined with small beads (e.g., glass, steel, or ceramic beads)that are mixed (e.g., vortexed) with the cell suspension at high speeds.The beads collide with the cells, breaking open the cell membrane withshear forces. After “bead beating”, the cell suspension is centrifugedto pellet the cellular debris and beads, and the supernatant may bepurified via a nucleic acid binding column (such as the MagMAX™Viral/Pathogen Nucleic Acid Isolation Kit from ThermoFisher (Waltham,MA, USA) and others from Qiagen (Hilden, Germany), TakaraBio (San Jose,CA, USA), and Biocomma (Shenzen, China)) to collect the nucleic acids(see the discussion of solid phase extraction below).

Solid phase extraction (SPE): Another method for capturing nucleic acidsis through solid phase extraction. SPE involves a liquid and stationaryphase, which selectively separate the target analyte (here, nucleicacids) from the liquid in which the cells are suspended based onspecific hydrophobic, polar, and/or ionic properties of the targetanalyte in the liquid and the stationary solid matrix. Silica bindingcolumns and their derivatives are the most commonly used SPE techniques,having a high binding affinity for DNA under alkaline conditions andincreased salt concentration; thus, a highly alkaline and concentratedsalt buffer is used. The nucleic acid sample is centrifuged through acolumn with a highly porous and high surface area silica matrix, wherebinding occurs via the affinity between negatively charged nucleic acidsand positively charged silica material. The nucleic acids bind to thesilica matrices, while the other cell components and chemicals passthrough the matrix without binding. One or more wash steps typically areperformed after the initial sample binding (i.e., the nucleic acids tothe matrix), to further purify the bound nucleic acids, removing excesschemicals and cellular components non-specifically bound to the silicamatrix. Alternative versions of SPE include reverse SPE and ion exchangeSPE, and use of glass particles, cellulose matrices, and magnetic beads.

Thermal lysis: Thermal lysis involves heating a sample of mammaliancells, virions, or bacterial cells at high temperatures thereby damagingthe cellular membranes by denaturizing the membrane proteins.Denaturizing the membrane proteins results in the release ofintracellular DNA. Cells are generally heated above 90° C., however timeand temperature may vary depending on sample volume and sample type.Once lysed, typically one or more downstream methods, such as use ofnucleic acid binding columns for solid phase extraction as describedabove, are required to further purify the nucleic acids.

Physical lysis: Common physical lysis methods include sonication andosmotic shock. Sonication involves creating and rupturing of cavities orbubbles to release shockwaves, thereby disintegrating the cellularmembranes of the cells. In the sonication process, cells are added intolysis buffer, often containing phenylmethylsulfonyl fluoride, to inhibitproteases. The cell samples are then placed in a water bath and asonication wand is placed directly into the sample solution. Sonicationtypically occurs between 20-50 kHz, causing cavities to be formedthroughout the solution as a result of the ultrasonic vibrations;subsequent reduction of pressure then causes the collapse of the cavityor bubble resulting in a large amount of mechanical energy beingreleased in the form of a shockwave that propagates through the solutionand disintegrates the cellular membrane. The duration of the sonicationpulses and number of pulses performed varies depending on cell type andthe downstream application. After sonication, the cell suspensiontypically is centrifuged to pellet the cellular debris and thesupernatant containing the nucleic acids may be further purified bysolid phase extraction as described above.

Another form of physical lysis is osmotic shock, which is most typicallyused with mammalian cells. Osmotic shock involves placing cells inDI/distilled water with no salt added. Because the salt concentration islower in the solution than in the cells, water is forced into the cellcausing the cell to burst, thereby rupturing the cellular membrane. Thesample is typically purified and extracted by techniques such as e.g.,solid phase extraction or other techniques known to those of skill inthe art.

Chemical lysis: Chemical lysis involves rupturing cellular and nuclearmembranes by disrupting the hydrophobic-hydrophilic interactions in themembrane bilayers via detergents. Salts and buffers (such as, e.g.,Tris-HCl pH8) are used to stabilize pH during extraction, and chelatingagents (such as ethylenediaminetetraacetic acid (EDTA)) and inhibitors(e.g., Proteinase K) are also added to preserve the integrity of thenucleic acids and protect against degradation. Often, chemical lysis isused with enzymatic disruption methods (see below) for lysing bacterialcell walls. In addition, detergents are used to lyse and break downcellular membranes by solubilizing the lipids and membrane proteins onthe surface of cells. The contents of the cells include, in addition tothe desired nucleic acids, inner cellular proteins and cellular debris.Enzymes and other inhibitors are added after lysis to inactivatenucleases that may degrade the nucleic acids. Proteinase K is commonlyadded after lysis, destroying DNase and RNase enzymes capable ofdegrading the nucleic acids. After treatment with enzymes, the sample iscentrifuged, pelleting cellular debris, while the nucleic acids remainin the solution. The nucleic acids may be further purified as describedabove.

Another form of chemical lysis is the widely-used procedure ofphenol-chloroform extraction. Phenol-chloroform extraction involves theability for nucleic acids to remain soluble in an aqueous solution in anacidic environment, while the proteins and cellular debris can bepelleted down via centrifugation. Phenol and chloroform ensure a clearseparation of the aqueous and organic (debris) phases. For DNA, a pH of7-8 is used, and for RNA, a more acidic pH of 4.5 is used.

Enzymatic lysis: Enzymatic disruption methods are commonly combined withother lysis methods such as those described above to disrupt cellularwalls (bacteria and plants) and membranes. Enzymes such as lysozyme,lysostaphin, zymolase, and protease are often used in combination withother techniques such as physical and chemical lysis. For example, onecan use cellulase to disrupt plant cell walls, lysosomes to disruptbacterial cell walls and zymolase to disrupt yeast cell walls.

Example II: RNP Formation

For RNP complex formation, 250 nM of LbCas12a nuclease protein wasincubated with 375 nM of a target specific gRNA in 1× Buffer (10 mMTris-HCl, 100 μg/mL BSA) with 2-15 mM MgCl₂ at 25° C. for 20 minutes.The total reaction volume was 2 μL. Other ratios of LbCas12a nuclease togRNAs were tested, including 1:1, 1:2 and 1:5. The incubationtemperature can range from 20° C.-37° C., and the incubation time canrange from 10 minutes to 4 hours.

Example III: Blocked Nucleic Acid Molecule Formation

Ramp cooling: For formation of the secondary structure of blockednucleic acids, 2.5 μM of a blocked nucleic acid molecule (any ofFormulas I-N) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl)with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes todehybridize any secondary structures. Thereafter, the reaction wascooled to 37° C. at 0.015° C./second to form the desired secondarystructure.

Snap cooling: For formation of the secondary structure of blockednucleic acids, 2.5 μM of a blocked nucleic acid molecule (any ofFormulas I-N) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl)with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes todehybridize any secondary structures. Thereafter, the reaction wascooled to room temperature by removing the heat source to form thedesired secondary structure.

Snap cooling on ice: For formation of the secondary structure of blockednucleic acids, 2.5 μM of a blocked nucleic acid molecule (any ofFormulas I-N) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl)with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes todehybridize any secondary structures. Thereafter, the reaction wascooled to room temperature by placing the reaction tube on ice to formthe desired secondary structure.

Example IV: Reporter Moiety Formation

The reporter moieties used in the reactions herein were single-strandedDNA oligonucleotides 5-10 bases in length (e.g., with sequences ofTTATT, TTTATTT, ATTAT, ATTTATTTA, AAAAA, or AAAAAAAAA) with afluorophore and a quencher attached on the 5′ and 3′ ends, respectively.In one example using a Cas12a cascade, the fluorophore was FAM-6, andthe quencher was IOWA BLACK® (Integrated DNA Technologies, Coralville,IA). In another example using a Cas13 cascade, the reporter moietieswere single stranded RNA oligonucleotides 5-10 bases in length (e.g.,r(U)n, r(UUAUU)n, r(A)n).

Example V: Cascade Assay

First Format (final reaction mixture components added at the same time):RNP1 was assembled using the LbCas12a nuclease and a gRNA for theMethicillin resistant Staphylococcus aureus (MRSA) DNA according to theRNP complex formation protocol described in Example II (for thissequence, see Example VIII). Briefly, 250 nM LbCas12a nuclease wasassembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 wasformed using the LbCas12a nuclease and a gRNA specific for a selectedblocked nucleic acid molecule (Formula I-N) using 500 nM LbCas12anuclease assembled with 750 nM of the blocked nucleic acid-specific gRNAincubated in 1×NEB 2.1 Buffer (New England Biolabs, Ipswich, MA) with 5mM MgCl₂ at 25° C. for 20-40 minutes. Following incubation, RNPIs werediluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. Thereafter,the final reaction was carried out in 1× Buffer, with 500 nM of thessDNA reporter moiety, 1×ROX dye (Thermo Fisher Scientific, Waltham, MA)for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, 15 nM LbCas12a: 22.5 nMgRNA RNP1, 20 nM LbCas12a: 35 nM gRNA RNP2, and 50 nM blocked nucleicacid molecule (any one of Formula I-N) in a total volume of 9 μL. 1 μLof MRSA DNA target (with samples having as low as three copies and asmany as 30000 copies—see FIGS. 6-11 ) was added to make a final volumeof 10 μL. The final reaction was incubated in a thermocycler at 25° C.with fluorescence measurements taken every 1 minute.

Second Format (RNP1 and MRSA target pre-incubated before addition tofinal reaction mixture): RNP1 was assembled using the LbCas12a nucleaseand a gRNA for the MRSA DNA according to RNP formation protocoldescribed in Example II (for this sequence, see Example VIII). Briefly,250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-targetspecific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and agRNA specific for a selected blocked nucleic acid molecule (FormulaI-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of theblocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (NewEngland Biolabs, Ipswich, MA) with 5 mM MgCl₂ at 25° C. for 20-40minutes. Following incubation, RNPIs were diluted to a concentration of75 nM LbCas12a: 112.5 nM gRNA. After dilution, the formed RNP1 was mixedwith 1 μL of MRSA DNA target and incubated at 20° C.-37° C. for up to 10minutes to activate RNP1. The final reaction was carried out in 1×Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (ThermoFisher Scientific, Waltham, MA) for passive reference, 2.5 mM MgCl₂, 4mM NaCl, the pre-incubated and activated RNP1, 20 nM LbCas12a: 35 nMgRNA RNP2, and 50 nM blocked nucleic acid molecule (any one of FormulaI-N) in a total volume of 9 μL. The final reaction was incubated in athermocycler at 25° C. with fluorescence measurements taken every 1minute.

Third Format (RNP1 and MRSA target pre-incubated before addition tofinal reaction mixture and blocked nucleic acid molecule added to finalreaction mixture last): RNP1 was assembled using the LbCas12a nucleaseand a gRNA for the MRSA DNA according to the RNP complex formationprotocol described in Example II (for this sequence, see Example VIII).Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of theMRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12anuclease and a gRNA specific for a selected blocked nucleic acidmolecule (Formula I-IV) using 500 nM LbCas12a nuclease assembled with750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB 2.1Buffer (New England Biolabs, Ipswich, MA) with 5 mM MgCl₂ at 25° C. for20-40 minutes. Following incubation, RNPIs were diluted to aconcentration of 75 nM LbCas12a: 112.5 nM gRNA. After dilution, theformed RNP1 was mixed with 1 μL of MRSA DNA target and incubated at 20°C.-37° C. for up to 10 minutes to activate RNP1. The final reaction wascarried out in 1× Buffer, with 500 nM of the ssDNA reporter moiety,1×ROX dye (Thermo Fisher Scientific, Waltham, MA) for passive reference,2.5 mM MgCl₂, 4 mM NaCl, the pre-incubated and activated RNP1, and 20 nMLbCas12a: 35 nM gRNA RNP2 in a total volume of 9 μL. Once the reactionmixture was made, 1 μL (50 nM) blocked nucleic acid molecule (any one ofFormula I-N) was added for a total volume of 10 μL. The final reactionwas incubated in a thermocycler at 25° C. with fluorescence measurementstaken every 1 minute.

Example VI: Detection of MRSA and Test Reaction Conditions

To detect the presence of Methicillin resistant Staphylococcus aureus(MRSA) and determine the sensitivity of detection with the cascadeassay, titration experiments with a MRSA DNA target nucleic acid ofinterest were performed. The MRSA mecA gene DNA sequence (NCBI ReferenceSequence NC: 007793.1) is as follows.

SEQ ID NO: 1: ATGAAAAAGATAAAAATTGTTCCACTTATTTTAATAGTTGTAGTTGTCGGGTTTGGTATATATTTTTATGCTTCAAAAGATAAAGAAATTAATAATACTATTGATGCAATTGAAGATAAAAATTTCAAACAAGTTTATAAAGATAGCAGTTATATTTCTAAAAGCGATAATGGTGAAGTAGAAATGACTGAACGTCCGATAAAAATATATAATAGTTTAGGCGTTAAAGATATAAACATTCAGGATCGTAAAATAAAAAAAGTATCTAAAAATAAAAAACGAGTAGATGCTCAATATAAAATTAAAACAAACTACGGTAACATTGATCGCAACGTTCAATTTAATTTTGTTAAAGAAGATGGTATGTGGAAGTTAGATTGGGATCATAGCGTCATTATTCCAGGAATGCAGAAAGACCAAAGCATACATATTGAAAATTTAAAATCAGAACGTGGTAAAATTTTAGACCGAAACAATGTGGAATTGGCCAATACAGGAACAGCATATGAGATAGGCATCGTTCCAAAGAATGTATCTAAAAAAGATTATAAAGCAATCGCTAAAGAACTAAGTATTTCTGAAGACTATATCAAACAACAAATGGATCAAAATTGGGTACAAGATGATACCTTCGTTCCACTTAAAACCGTTAAAAAAATGGATGAATATTTAAGTGATTTCGCAAAAAAATTTCATCTTACAACTAATGAAACAGAAAGTCGTAACTATCCTCTAGGAAAAGCGACTTCACATCTATTAGGTTATGTTGGTCCCATTAACTCTGAAGAATTAAAACAAAAAGAATATAAAGGCTATAAAGATGATGCAGTTATTGGTAAAAAGGGACTCGAAAAACTTTACGATAAAAAGCTCCAACATGAAGATGGCTATCGTGTCACAATCGTTGACGATAATAGCAATACAATCGCACATACATTAATAGAGAAAAAGAAAAAAGATGGCAAAGATATTCAACTAACTATTGATGCTAAAGTTCAAAAGAGTATTTATAACAACATGAAAAATGATTATGGCTCAGGTACTGCTATCCACCCTCAAACAGGTGAATTATTAGCACTTGTAAGCACACCTTCATATGACGTCTATCCATTTATGTATGGCATGAGTAACGAAGAATATAATAAATTAACCGAAGATAAAAAAGAACCTCTGCTCAACAAGTTCCAGATTACAACTTCACCAGGTTCAACTCAAAAAATATTAACAGCAATGATTGGGTTAAATAACAAAACATTAGACGATAAAACAAGTTATAAAATCGATGGTAAAGGTTGGCAAAAAGATAAATCTTGGGGTGGTTACAACGTTACAAGATATGAAGTGGTAAATGGTAATATCGACTTAAAACAAGCAATAGAATCATCAGATAACATTTTCTTTGCTAGAGTAGCACTCGAATTAGGCAGTAAGAAATTTGAAAAAGGCATGAAAAAACTAGGTGTTGGTGAAGATATACCAAGTGATTATCCATTTTATAATGCTCAAATTTCAAACAAAAATTTAGATAATGAAATATTATTAGCTGATTCAGGTTACGGACAAGGTGAAATACTGATTAACCCAGTACAGATCCTTTCAATCTATAGCGCATTAGAAAATAATGGCAATATTAACGCACCTCACTTATTAAAAGACACGAAAAACAAAGTTTGGAAGAAAAATATTATTTCCAAAGAAAATATCAATCTATTAACTGATGGTATGCAACAAGTCGTAAATAAAACACATAAAGAAGATATTTATAGATCTTATGCAAACTTAATTGGCAAATCCGGTACTGCAGAACTCAAAATGAAACAAGGAGAAACTGGCAGACAAATTGGGTGGTTTATATCATATGATAAAGATAATCCAAACATGATGATGGCTATTAATGTTAAAGATGTACAAGATAAAGGAATGGCTAGCTACAATGCCAAAATCTCAGGTAAAGTGTATGATGAGCTATATGAGAACGGTAATAAAAAATACGATATAGA TGAATAA

Briefly, an RNP1 was preassembled with a gRNA sequence designed totarget MRSA DNA. Specifically, RNP1 was designed to target a 20 bpregion of the mecA gene of MRSA: TGTATGGCATGAGTAACGAA (SEQ ID NO: 2). AnRNP2 was preassembled with a gRNA sequence designed to target theunblocked nucleic acid molecule that results from unblocking (i.e.,linearizing) blocked nucleic acid molecule U29 (FIG. 6A). The reactionmixture contained the preassembled RNP1, preassembled RNP2, and ablocked nucleic acid molecule, in a buffer (pH of about 8) containing 4mM MgCl₂ and 101 mM NaCl and the reaction was performed at 25° C.

As stated above, the present disclosure describes controlling reactionkinetics in a cascade assay via molecular design of one of the assaycomponents, the blocked nucleic acid molecule or the blocked primermolecule that serves as the target molecule of RNP2. As shown below,stronger regions of hybridization (or self-hybridization) via bothlength and GC content—leads to slower kinetics; that is, the morenegative the Gibbs free energy of the blocked nucleic acid molecule orblocked primer molecule, the slower the reaction kinetics.

FIG. 6A shows the structure and segment parameters of molecule U29. Notemolecule U29 has a secondary structure Gibbs free energy value of −5.85kcal/mol and relatively short self-hybridizing, double stranded regions(“clamps”) of 5 bases and 6 bases. FIGS. 6B-6H show the results achievedfor detection of 3E4 copies, 30 copies, 3 copies and 0 copies of themecA gene of MRSA (n=3) at 25° C. with varying concentrations of blockednucleic acid, RNP2 and reporter moiety. FIG. 6B shows the resultsachieved when 100 nM blocked nucleic acid molecules, 10 nM RNP2s and 500nM reporter moieties are used. Thus, in this experiment, the ratio ofblocked nucleic acid molecules to RNP2s is 10:1. Note first that with 3Mcopies, nearly 100% of the reporters are cleaved at t=1 with asignal-to-noise ratio of 28.06 at 0 minutes, a signal-to-noise ratio of24.23 at 5 minutes, and a signal-to-noise ratio of 21.01 at 10 minutes.Additionally, the signal-to-noise ratios for detection with 30 copies ofMRSA target is 12.45 at 0 minutes, 14.07 at 5 minutes and 16.16 at 10minutes; and the signal-to-noise ratios for detection with 3 copies ofMRSA target is 1.79 at 0 minutes, 1.64 at 5 minutes and is 2.04 at 10minutes. Note the measured fluorescence for 0 copies of MRSA targetincreases only slightly over the 10- and 30-minutes intervals, resultingin a flat negative. A flat negative signal (the results obtained overthe time period for 0 copies) demonstrates that there is very littleundesired signal generation in the system. Note that the negative signalwhen the ratio of blocked nucleic acid molecules to RNP2s is 10:1 isflatter than those in FIGS. 6C through 6H.

FIG. 6C shows the results achieved when 50 nM blocked nucleic acidmolecules, 10 nM RNP2s and 500 nM reporter moieties are used. Thus, inthis experiment, the ratio of blocked nucleic acid molecules to RNP2s is5:1. Note first that with 3E4 copies, again nearly 100% of the reportersare cleaved at t=1 with a signal-to-noise ratio of 12.85, asignal-to-noise ratio of 10.51 at 5 minutes, and a signal-to-noise ratioof 8.18 at 10 minutes. Additionally, the signal-to-noise ratios fordetection with 30 copies of MRSA target is 5.85 at 0 minutes, 6.44 at 5minutes and 6.48 at 10 minutes; and the signal-to-noise ratios fordetection with 3 copies of MRSA target is 1.54 at 0 minutes, 1.61 at 5minutes and is 1.71 at 10 minutes. Note the measured fluorescence at 0copies of MRSA target increases, resulting a less flat negative than the10:1 ratio of blocked nucleic acid molecules to RNP2.

FIG. 6D shows the results achieved when 50 nM blocked nucleic acidmolecules, 10 nM RNP2s and 2500 nM reporter moieties are used. Thus, inthis experiment, the ratio of blocked nucleic acid molecules to RNP2s is5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved att=1 with a signal-to-noise ratio of 34.92, a signal-to-noise ratio of30.62 at 5 minutes, and a signal-to-noise ratio of 25.81 at 10 minutes.Additionally, the signal-to-noise ratios for detection with 30 copies ofMRSA target is 7.97 at 0 minutes, 1.73 at 5 minutes and 10.50 at 10minutes; and the signal-to-noise ratios for detection with 3 copies ofMRSA target is 1.65 at 0 minutes, 1.73 at 5 minutes and is 1.82 at 10minutes. Note the measured fluorescence at 0 copies of MRSA targetincreases, resulting in a less flat negative than the 10:1 ratio ofblocked nucleic acid molecules to RNP2s, but possibly due to the 5×increase in the concentration of reporter moieties; however, note alsothat a higher concentration of reporter moieties allows for a highersignal-to-noise ratio for 3E4 and 30 copies of MRSA target.

FIG. 6E shows the results achieved when 100 nM blocked nucleic acidmolecules, 20 nM RNP2s and 500 nM reporter moieties are used and 4 mMNaCl. Thus, in this experiment, the ratio of blocked nucleic acidmolecules to RNP2s is 5:1 but double the concentration of both of thesemolecules than that shown in FIGS. 6C and 6D. With 3M copies, againnearly 100% of the reporters are cleaved at t=1 with a signal-to-noiseratio of 11.89, a signal-to-noise ratio of 8.97 at 5 minutes, and asignal-to-noise ratio of 6.53 at 10 minutes. Additionally, thesignal-to-noise ratios for detection with 30 copies of MRSA target is5.46 at 0 minutes, 5.85 at 5 minutes and 5.43 at 10 minutes; and thesignal-to-noise ratios for detection with 3 copies of MRSA target is1.58 at 0 minutes, 1.65 at 5 minutes and is 1.80 at 10 minutes. Note themeasured fluorescence at 0 copies of MRSA increases, resulting in a lessflat negative than the 10:1 ratio of blocked nucleic acid molecules toRNP2s shown in FIG. 6B. Note also that the ratio of blocked nucleic acidmolecules to RNP2s (5:1) appears to be more important than the ultimateconcentration (100 nM/20 nM) by comparison to FIG. 6D where the ratio ofblocked nucleic acid molecules to RNP2s was also 5:1; however, theconcentration of blocked nucleic acid molecules was 50 nM and theconcentration of RNP2 was 10 nM.

FIG. 6F shows the results achieved when 50 nM blocked nucleic acidmolecules, 20 nM RNP2s and 500 nM reporter moieties are used and using aconcentration of 4 mM NaCl. In this experiment the ratio of blockednucleic acid molecules to RNP2s is 2.5:1. With 3E4 copies, again nearly100% of the reporters are cleaved at t=1 with a signal-to-noise ratio of25.85, a signal-to-noise ratio of 21.36 at 5 minutes, and asignal-to-noise ratio of 16.24 at 10 minutes. Additionally, thesignal-to-noise ratios for detection with 30 copies of MRSA target is5.28 at 0 minutes, 6.19 at 5 minutes and 7.02 at 10 minutes; and thesignal-to-noise ratios for detection with 3 copies of MRSA target isvery low at 0 minutes, 1.53 at 5 minutes and is 1.73 at 10 minutes. Notethe measured fluorescence at 0 copies of MRSA target increases,resulting in a less flat negative than the 10:1 ratio of blocked nucleicacid molecules to RNP2s shown in FIG. 6B. Note also that thesignal-to-noise ratio for all concentrations was reduced at the 2.5:1ratio of blocked nucleic acid molecules to RNP2s.

FIG. 6G shows the results achieved when 50 nM blocked nucleic acidmolecules, 20 nM RNP2s and 500 nM reporter moieties are used and using aconcentration of 10 mM NaCl. Thus, in this experiment, the ratio ofblocked nucleic acid molecules to RNP2s is 2.5:1. With 3E4 copies, againnearly 100% of the reporters are cleaved at t=1 with a signal-to-noiseratio of 12.75, a signal-to-noise ratio of 7.78 at 5 minutes, and asignal-to-noise ratio of 3.66 at 10 minutes. Additionally, thesignal-to-noise ratios for detection with 30 copies of MRSA target is6.09 at 0 minutes, 6.23 at 5 minutes and 3.58 at 10 minutes; and thesignal-to-noise ratios for detection with 3 copies of MRSA target isvery low at 0 minutes, 1.40 at 5 minutes and is 1.62 at 10 minutes. Notethe measured fluorescence at 0 copies increases, resulting in less of aflat negative than the 10:1 ratio of blocked nucleic acid molecules toRNP2s shown in FIG. 6B. Note also that the signal-to-noise ratio for allconcentrations was reduced substantially at the 2.5:1 ratio of blockednucleic acid molecules to RNP2s and that the NaCl concentration at 10 mMvs. 4 mM (FIG. 6F) did not make much of a difference.

FIG. 6H shows the results achieved when 100 nM blocked nucleic acidmolecules, 20 nM RNP2s and 500 nM reporter moieties are used and using aconcentration of 10 mM NaCl. Thus, in this experiment, the ratio ofblocked nucleic acid molecules to RNP2s is 5:1. With 3E4 copies, againnearly 100% of the reporters are cleaved at t=1 with a signal-to-noiseratio of 77.38, a signal-to-noise ratio of 74.18 at 5 minutes, and asignal-to-noise ratio of 67.90 at 10 minutes. Additionally, thesignal-to-noise ratios for detection with 30 copies of MRSA target is5.94 at 0 minutes, 7,45 at 5 minutes and 9.73 at 10 minutes; and thesignal-to-noise ratios for detection with 3 copies of MRSA target is1.66 at 0 minutes, 2.13 at 5 minutes and is 2.38 at 10 minutes. Note themeasured fluorescence at 0 copies of MRSA target increases slightly,resulting in a less flat negative than the 10:1 ratio of blocked nucleicacid molecules to RNP2s shown in FIG. 6B. Note also that thesignal-to-noise ratio for all concentrations was increased substantiallyat the 5:1 ratio of blocked nucleic acid molecules to RNP2s as comparedto the 2.5:1 ratio of blocked nucleic acid molecules to RNP2s. Insummary, the results shown in FIGS. 6B-6H indicate that a 5:1 ratio ofblocked nucleic acid molecules to RNP2s or greater leads to highersignal-to-noise ratios for all concentrations of MRSA target.

FIG. 7A shows the structure and segment parameters of molecule F375.Note molecule F375 has a secondary structure Gibbs free energy value of−14.50 kcal/mol with longer clamps (7 bases and 7 bases) relative tomolecule U29 (5 bases and 6 bases). FIG. 7B shows the results achievedfor detection of 3E4 copies, 30 copies, 3 copies and 0 copies of themecA gene of MRSA (n=3) at 25° C. at 10 or 30 minutes as indicated,where 50 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nMreporter moieties are used. Thus, in this experiment, the ratio ofblocked nucleic acid molecules to RNP2s is 5:1. For 3E4 copies of MRSAtarget, a signal-to-noise ratio of 21.82 is achieved at 0 minutes, asignal-to-noise ratio of 43.81 is achieved at 5 minutes, and asignal-to-noise ratio of 56.19 is achieved at 10 minutes. Additionally,the signal-to-noise ratios for detection with 30 copies of MRSA is 3.99at 0 minutes 7.44 at 5 minutes and 11.55 at 10 minutes. Thesignal-to-noise ratio for detection with 3 copies of MRSA is nearly 1 at0 minutes, 1.95 at 10 minutes and is 2.43 at 30 minutes. Note that thereaction kinetics for molecule F375 vs. U29 are much slower. (Note thatFIG. 6C shows the results for U29 with comparable conditions.) For U29at t=1 and 3e4 copies of MRSA target, almost 100% of the reportermolecules are cleaved but not so with F375. Also, for both 30 copies and3 copies of MRSA target, the U29 blocked nucleic acid molecule has muchhigher fluorescence at t=1 than F375.

FIG. 8A shows the structure and segment parameters of molecule U250.Note molecule U250 has a secondary structure Gibbs free energy value of−9.01 kcal/mol with longer clamp regions (7 bases and 7 bases) than U29(5 bases and 6 bases) but equal-sized clamp regions of F375 (7 bases and7 bases), but that U250 has a larger loop region (11 bases vs. 7 bases).FIG. 8B shows the results achieved for detection of 3M copies, 30copies, 3 copies and 0 copies of the mecA gene of MRSA (n=3) at 25° C.at 10 or 30 minutes as indicated, where 50 nM blocked nucleic acidmolecules, 20 nM RNP2s and 500 nM reporter moieties are used. Thus, inthis experiment, the ratio of blocked nucleic acid molecules to RNP2s is5:1. For 3M copies, a signal-to-noise ratio of 10.37 is achieved at 0minutes, a signal-to-noise ratio of 17.70 is achieved at 5 minutes, anda signal-to-noise ratio of 28.62 is achieved at 10 minutes.Additionally, the signal-to-noise ratios for detection with 30 copies ofMRSA target is 6.49 at 0 minutes 7.99 at 5 minutes and 10.33 at 10minutes. The signal-to-noise ratio for detection with 3 copies of MRSAtarget is nearly 1 at 0 minutes, 3.45 at 10 minutes and is 3.46 at 30minutes. Note that the reaction kinetics for U250 are similar to thoseof molecule F375, and far slower than those for U29. (Note that FIG. 6Cshows the results for U29 with comparable conditions.) For U29 at t=1and 3M copies of MRSA target, almost 100% of the reporter molecules arecleaved but not so with F375. For both 30 copies and 3 copies of MRSAtarget, the U29 blocked nucleic acid molecule has much higherfluorescence at t=1 than F375.

FIG. 9A shows the structure and segment parameters of molecule T135.Note molecule T135 has a secondary structure Gibbs free energy value of−17.60 kcal/mol with longer clamp regions (10 bases and 12 bases) thanall of U29, F375 and U250. FIG. 9B shows the results achieved fordetection of 3M copies, 30 copies, 3 copies and 0 copies of the mecAgene of MRSA (n=3) at 25° C. at 10 or 30 minutes as indicated, where 50nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reportermoieties are used. Thus, in this experiment as in the others where theresults are shown in FIGS. 6B, 7B and 8B, the ratio of blocked nucleicacid molecules to RNP2s is 5:1. For 3M copies, a signal-to-noise ratioof 42.74 is achieved at 0 minutes, a signal-to-noise ratio of 106.61 isachieved at 5 minutes, and a signal-to-noise ratio of 91.04 is achievedat 10 minutes. Additionally, the signal-to-noise ratios for detectionwith 30 copies of MRSA target is 9.75 at 0 minutes, 15.77 at 5 minutesand 20.04 at 10 minutes. The signal-to-noise ratio for detection with 3copies of MRSA target is nearly 1 at 0 minutes, 3.18 at 5 minutes, 3.07at 10 minutes and is 3.64 at 30 minutes. Note that the reaction kineticsfor T135 is similar to those for F375 and U250, but much slower than thereaction kinetics for U29. Note also that the signal-to-noise ratio for3M and 30 copies of MRSA target was very high.

FIG. 10A shows the structure and segment parameters of molecule T134.Note molecule T134 has a secondary structure Gibbs free energy value of−16.13 kcal/mol with clamp regions of 9 bases and 12 bases, on par withthose of molecule T135. FIG. 10B shows the results achieved fordetection of 3E4 copies, 30 copies, 3 copies and 0 copies of the mecAgene of MRSA (n=3) at 25° C. at 10 or 30 minutes as indicated, where 50nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reportermoieties are used. Thus, in this experiment as in the others where theresults are shown in FIGS. 6B, 7B, 8B, and 10B the ratio of blockednucleic acid molecules to RNP2s is 5:1. For 3E4 copies, asignal-to-noise ratio of 63.06 is achieved at 0 minutes, asignal-to-noise ratio of 194 is achieved at 5 minutes, and asignal-to-noise ratio of 194 is achieved at 10 minutes. Additionally,the signal-to-noise ratios for detection with 30 copies of MRSA targetis 13.99 at 0 minutes, 32.23 at 5 minutes and 26.82 at 10 minutes. Thesignal-to-noise ratio for detection with 3 copies of MRSA target isnearly 1 at 0 minutes, 2.34 at 10 minutes and is 2.44 at 30 minutes. Thereaction kinetics for molecule T134 is roughly comparable to thereaction kinetics for molecule T135; also with exceptionalsignal-to-noise ratios at 10 minutes, and even at t=1 and t=5.

FIG. 11A shows the structure and segment parameters of molecule T119.Note molecule T119 has a secondary structure Gibbs free energy value of−17.53 kcal/mol with longer clamp regions (10 bases and 12 bases) thanall of U29, F375, U250, T135 and T134. FIG. 11B shows the resultsachieved for detection of 3E4 copies, 30 copies, 3 copies and 0 copiesof the mecA gene of MRSA (n=3) at 25° C. at 10 or 30 minutes asindicated, where 50 nM blocked nucleic acid molecules, 20 nM RNP2s and500 nM reporter moieties are used. Thus, in this experiment as in theothers where the results are shown in FIGS. 6B, 7B, 8B, 9B, and 10B theratio of blocked nucleic acid molecules to RNP2s is 5:1. For 3M copies,a signal-to-noise ratio of 21.27 is achieved at 0 minutes, asignal-to-noise ratio of 69.55 is achieved at 5 minutes, and asignal-to-noise ratio of 231 is achieved at 10 minutes. Additionally,the signal-to-noise ratios for detection with 30 copies of MRSA is 4.56at 0 minutes, 10.88 at 5 minutes and 25.66 at 10 minutes. Thesignal-to-noise ratio for detection with 3 copies of MRSA is nearly 1 at0 minutes, 7.09 at 10 minutes and is 3.36 at 30 minutes. The kineticsfor molecule T119 are similar to those of T134, also with exceptionalsignal-to-noise ratios at t=1, t=5 and t=10.

In summary, different designs with different clamps allow for differentdetection kinetics; that is, the kinetics of the detection reaction canbe controlled by the design of the blocked nucleic acid molecule; thus,the blocked nucleic acid molecules are quantitative or semi-quantitativeby design. Very long clamps lead to longer detection times but alsoallow for higher resolution for quantifying target nucleic acids ofinterest over the longer time period. Note again that the cascade assayreactions were carried out at 25° C.

While certain embodiments have been described, these embodiments havebeen presented by way of example only and are not intended to limit thescope of the present disclosures. Indeed, the novel methods,apparatuses, modules, instruments and systems described herein can beembodied in a variety of other forms; furthermore, various omissions,substitutions and changes in the form of the methods, apparatuses,modules, instruments and systems described herein can be made withoutdeparting from the spirit of the present disclosures. The accompanyingclaims and their equivalents are intended to cover such forms ormodifications as would fall within the scope and spirit of the presentdisclosures.

PARTIES TO A JOINT RESEARCH AGREEMENT

The presently claimed invention was made by or on behalf of thebelow-listed parties to a joint research agreement. The joint researchagreement was in effect on or before the date the claimed invention wasmade, and the claimed invention was part of the joint research agreementand made as a result of activities undertaken within the scope of thejoint research agreement. The parties to the joint research agreementare The Board of Trustees of the University of Illinois and LabSimply,Inc. (now VedaBio, Inc.).

We claim:
 1. A composition of matter for detecting a nucleic acid targetof interest in a sample comprising: first ribonucleoprotein complexes(RNP1s), wherein the RNP1s comprise a first nucleic acid-guided nucleaseand a first gRNA; wherein the first gRNA comprises a sequencecomplementary to the nucleic acid target of interest, and wherein thefirst nucleic acid-guided nuclease exhibits both cis-cleavage activityand trans-cleavage activity; second ribonucleoprotein complexes (RNP2s),wherein the RNP2s comprise a second nucleic acid-guided nuclease and asecond gRNA that is not complementary to the nucleic acid target ofinterest, and wherein the second nucleic acid-guided nuclease exhibitsboth cis- and trans-cleavage activity; and a plurality of tunableblocked nucleic acid molecules, wherein the tunable blocked nucleic acidmolecules comprise: a first region recognized by the RNP2 complex; oneor more second regions not complementary to the first region forming atleast one loop; and one or more third regions complementary to andhybridized to the first region forming at least one clamp, wherein thefree energy of the plurality of tunable blocked primer molecules at 25°C. are at most about −5 kcal/mol when the following formula is used tocalculate the free energy for each base pair: ΔG° (T)=(ΔH°−TΔS°)calmol⁻¹, and total ΔG° is given by: ΔG°(total)=Σ_(i)n_(i) ΔG° (i)+ΔG°(init with term G·C)+ΔG° (init with term A·T)+ΔG° (sym), where ΔG° (i)are the standard free energy changes for the 10 possible Watson-CrickNNs, n_(i) is the number of occurrences of each nearest neighbor, i, andΔG° (sym) equals +0.43 kcal/mol if the duplex is self-complementary andzero if it is non-self-complementary, and wherein cleavage of the one ormore second regions results in dehybridization of the one or more thethird regions from the first region, resulting in an unblocked nucleicacid molecule.
 2. The composition of matter of claim 1, furthercomprising reporter moieties, wherein the reporter moieties produce adetectable signal upon trans-cleavage activity by the RNP2 to identifythe presence of one or more nucleic acid targets of interest in thesample.
 3. The composition of matter of claim 2, wherein the reportermoiety comprises a DNA, RNA or chimeric nucleic acid molecule and isoperably linked to the tunable blocked nucleic acid molecule.
 4. Thecomposition of matter of claim 3, wherein the detectable signal is afluorescent, chemiluminescent, radioactive, colorimetric or otheroptical signal.
 5. The composition of matter of claim 2, wherein thereporter moiety comprises a DNA, RNA or chimeric nucleic acid moleculeand is not operably linked to the tunable blocked nucleic acid molecule.6. The composition of matter of claim 5, wherein the detectable signalis a fluorescent, chemiluminescent, radioactive, colorimetric or otheroptical signal.
 7. The composition of matter of claim 1, wherein thetunable blocked nucleic acid molecule comprises a structure representedby any one of Formulas I-IV, wherein Formulas I-IV are in the 5′-to-3′direction: (a) A-(B-L)_(J)-C-M-T-D (Formula I); wherein A is 0-15nucleotides in length; B is 4-12 nucleotides in length; L is 3-25nucleotides in length; J is an integer between 1 and 10; C is 4-15nucleotides in length; M is 1-25 nucleotides in length or is absent,wherein if M is absent then A-(B-L)_(J)-C and T-D are separate nucleicacid strands; T is 17-135 nucleotides in length and comprises at least50% sequence complementarity to B and C; and D is 0-10 nucleotides inlength and comprises at least 50% sequence complementarity to A; (b)D-T-T′-C-(L-B)_(J)-A (Formula II); wherein D is 0-10 nucleotides inlength; T-T′ is 17-135 nucleotides in length; T is 1-10 nucleotides inlength and does not hybridize with T; C is 4-15 nucleotides in lengthand comprises at least 50% sequence complementarity to T; L is 3-25nucleotides in length and does not hybridize with T; B is 4-12nucleotides in length and comprises at least 50% sequencecomplementarity to T; J is an integer between 1 and 10; A is 0-15nucleotides in length and comprises at least 50% sequencecomplementarity to D; (c) T-D-M-A-(B-L)_(J)-C (Formula III); wherein Tis 17-135 nucleotides in length; D is 0-10 nucleotides in length; M is1-25 nucleotides in length or is absent, wherein if M is absent then T-Dand A-(B-L)_(J)-C are separate nucleic acid strands; A is 0-15nucleotides in length and comprises at least 50% sequencecomplementarity to D; B is 4-12 nucleotides in length and comprises atleast 50% sequence complementarity to T; L is 3-25 nucleotides inlength; J is an integer between 1 and 10; and C is 4-15 nucleotides inlength; or (d) T-D-M-A-L_(P)-C (Formula IV); wherein T is 17-31nucleotides in length (e.g., 17-100, 17-50, or 17-25); D is 0-15nucleotides in length; M is 1-25 nucleotides in length; A is 0-15nucleotides in length and comprises a sequence complementary to D; and Lis 3-25 nucleotides in length; p is 0or 1; C is 4-15 nucleotides inlength and comprises a sequence complementary to T.
 8. The compositionof matter of claim 7, wherein: (a) T of Formula I comprises at least 80%sequence complementarity to B and C; (b) D of Formula I comprises atleast 80% sequence complementarity to A; (c) C of Formula II comprisesat least 80% sequence complementarity to T; (d) B of Formula IIcomprises at least 80% sequence complementarity to T; (e) A of FormulaII comprises at least 80% sequence complementarity to D; (f) A ofFormula III comprises at least 80% sequence complementarity to D; (g) Bof Formula III comprises at least 80% sequence complementarity to T; (h)A of Formula IV comprises at least 80% sequence complementarity to D;and/or (i) C of Formula N comprises at least 80% sequencecomplementarity to T.
 9. The composition of matter of claim 1, whereinthe free energy of the tunable blocked nucleic acid molecule at 25° C.is at most about −5.5 kcal/mol and detection of the target nucleic acidof interest occurs instantaneously.
 10. The composition of matter ofclaim 9, wherein the free energy of the tunable blocked nucleic acidmolecule at 25° C. is at most about −10 kcal/mol.
 11. The composition ofmatter of claim 10, wherein the free energy of the tunable blockednucleic acid molecule at 25° C. is at most about −12 kcal/mol.
 12. Thecomposition of matter of claim 11, wherein the free energy of thetunable blocked nucleic acid molecule at 25° C. is at most about −17kcal/mol.
 13. The composition of matter of claim 1, wherein the freeenergy of the tunable blocked nucleic acid molecule at 25° C. has a freeenergy of about −5 kcal/mol to about −20 kcal/mol.
 14. The compositionof matter of claim 13, wherein the free energy of the tunable blockednucleic acid molecule at 25° C. has a free energy of about −13 kcal/molto about −20 kcal/mol.
 15. The composition of matter of claim 1, whereinthe tunable blocked nucleic acid molecule comprises at least 2 secondregions.
 16. The composition of matter of claim 15, wherein the tunableblocked nucleic acid molecule comprises at least 3 second regions. 17.The composition of matter of claim 1, wherein the tunable blockednucleic acid molecule comprises two separate but complementaryoligonucleotides.
 18. The composition of matter of claim 1, wherein thetunable blocked nucleic acid molecule comprises a single partiallyself-hybridizing oligonucleotide.
 19. The composition of matter of claim1, wherein the tunable nucleic acid nucleic acid molecule comprises amodified nucleoside or nucleotide.
 20. The composition of matter ofclaim 19, wherein the modified nucleoside or nucleotide comprises alocked nucleic acid (LNA), a peptide nucleic acid (PNA), a 2′-O-methyl(2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside,and/or a phosphorothioate (PS) bond.
 21. The composition of matter ofclaim 1, wherein the blocked nucleic acid molecule does not comprise aPAM sequence.
 22. The composition of matter of claim 1, wherein thetunable blocked nucleic acid molecule comprises a PAM sequence in theone or more second regions not complementary to the first region formingat least one loop.
 23. The composition of matter of claim 1, wherein theone or both of the RNP1 and the RNP2 comprise a nucleic acid-guidednuclease selected from Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e,Cas14, Cas12h, Cas12i, Cas12j, Cas13a, and Cas13b.
 24. The compositionof matter of claim 1, wherein the one or both of the RNP1 and the RNP2comprise a nucleic acid-guided nuclease that is a Type V nucleicacid-guided nuclease or a Type VI nucleic acid-guided nuclease.
 25. Thecomposition of matter of claim 1, wherein the first nucleic acid-guidednuclease is a different nucleic acid-guided nuclease than the secondnucleic acid-guided nuclease.
 26. The composition of matter of claim 1,wherein the first and/or second nucleic acid-guided nuclease comprises aRuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNHnuclease domain.
 27. The composition of matter of claim 1, whereinbinding of blocked nucleic acid molecules to RNP2 have high K_(d) valuesranging from about 100 nM to about 100 mM.
 28. The composition of matterof claim 1, wherein binding of unblocked nucleic acid molecules to RNP2have low K_(d) values ranging from about 100 fM to about 1 aM.
 29. Themethod of claim 1, wherein the K_(d) for the blocked nucleic acidmolecules about 10⁵-10¹⁰-fold or higher as compared to the K_(d) for theunblocked nucleic acid molecules.
 30. The composition of matter of claim1, wherein the composition includes 1 to about 1,000 different RNP1s.